MASS SPECTROMETRY

INTRODUCTION
Mass Spectrometer (MS) is a kind of machine which uses an analytical
technique to measure the mass-to charge ratio of ions. This analytical technique is also known as Mass
spectrometry. And an ion is an atom or group of atoms which have lost or gained one or more
electrons, making them negatively or positively charged. Mass spectrometry is an important emerging
method for the characterization of proteins. The two primary methods for ionization of whole proteins
are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). As it is an
important tool in proteomics, it is essential to understand not only the results, but also the principles of
Mass Spectrometer. This report is devoting to provide a simple but clear explanation to the principles
of Mass Spectrometer.

DEFINITION:
Mass spectrometry (MS) is an analytical technique for the determination of the
elemental composition of a sample or molecule. It is also used for elucidating the chemical structures
of molecules, such as peptides and other chemical compounds. The MS principle consists of ionizing
chemical compounds to generate charged molecules or molecule fragments and measurement of their
mass-to-charge ratios

PRINCIPLE:

In a typical MS procedure:

1. a sample is loaded onto the MS instrument, and

2. the components of the sample are ionized by one of a variety of methods (e.g., by impacting
them with an electron beam), which results in the formation of charged particles (ions)
3. directing the ions into an electric and/or magnetic field
4. computation of the mass-to-charge ratio of the particles based on the details of motion of the
ions as they transit through electromagnetic fields, and
5. Detection of the ions, which in step 4 were sorted according to m/z.
INSTRUMENTATION:
A mass spectrometer consists of following basic components
1. The inlet system (or) sample handling system
2. The ion source or ionisation chamber
3. The ion separator
4. The ion collector (the detector and readout system)
5. The vacuum system

THE MASS SPECTROMETER

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THE INLET SYSTEM:
• Direct Vapour Inlet.
• Gas Chromatography.
• Liquid Chromatography
• Direct Insertion Probe
• Direct Ionization of Sample

IONIZATION SOURCES
• Chemical Ionisation (CI)
• Atmospheric Pressure CI!(APCI)
• Electron Impact!(EI)
• Electrospray Ionization!(ESI)
• Matrix Assisted Laser Desorption Ionisation!(MALDI)
• Field Desorption/Field Ionisation (FD/FI)
• Fast Atom Bombardment (FAB)
• Thermo spray Ionisation (TI)

ANALYZERS
• quadruples
• Time-of-Flight (TOF)
• ion trap analyzer
• Fourier transform analyzer
• Orbitrap analyzer

DETECTORS
• electron multiplier detector
• Faraday cup detector
• Array detector

THE INLET SYSTEM/ SAMPLE HANDLING SYSTEM:

The selection of a sample inlet depends upon the sample and the sample matrix. Most ionization
techniques are designed for gas phase molecules so the inlet must transfer the analyte into the source
as a gas phase molecule. If the analyte is sufficiently volatile and thermally stable, a variety of inlets
are available. Gases and samples with high vapor pressure are introduced directly into the source
region. Liquids and solids are usually heated to increase the vapor pressure for analysis. If the analyte
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is thermally labile (it decomposes at high temperatures) or if it does not have a sufficient vapor
pressure, the sample must be directly ionized from the condensed phase. These direct ionization
techniques require special instrumentation and are more difficult to use. However, they greatly extend
the range of compounds that may be analyzed by mass spectrometry. Commercial instruments are
available that use direct ionization techniques to routinely analyze proteins and polymers with
molecular weights greater than 100,000 Dalton.

• DIRECT VAPOR INLET.

The simplest sample introduction method is a direct vapor inlet. The gas phase analyte is
introduced directly into the source region of the mass spectrometer through a needle valve. Pump
out lines are usually included to remove air from the sample. This inlet works well for gases,
liquids, or solids with a high vapor pressure. Samples with low vapour pressure are heated to
increase the vapor pressure. Since this inlet is limited to stable compounds and modest
temperatures, it only works for some samples.

• GAS CHROMATOGRAPHY.
Gas chromatography is probably the most common technique for introducing samples into a
mass spectrometer. Complex mixtures are routinely separated by gas chromatography and mass
spectrometry is used to identify and quantitate the individual components. Several different interface
designs are used to connect these two instruments. The most significant characteristics of the inlets are
the amount of GC carrier gas that enters the mass spectrometer and the amount of analyte that enters
the mass spectrometer. If a large flow of GC carrier gas enters the mass spectrometer it will increase
the pressure in the source region. Maintaining the required source pressure will require larger and more
expensive vacuum pumps. The amount of analyte that enters the mass spectrometer is important for
improving the detection limits of the instrument. Ideally all the analyte and none of the GC carrier gas
would enter the source region.

The most common GC/MS interface now uses a capillary GC column. Since the carrier Gas flow rate
is very small for these columns, the end of the capillary is inserted directly into the source region of the
mass spectrometer. The entire flow from the GC enters the mass spectrometer. Since capillary columns
are now very common, this inlet is widely used.

• LIQUID CHROMATOGRAPHY.
Liquid chromatography inlets are used to introduce thermally labile compounds not easily
separated by gas chromatography. These inlets have undergone considerable development and are now

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fairly routine. Because these inlets are used for temperature sensitive compounds, the sample is ionized
directly from the condensed phase.

• DIRECT INSERTION PROBE.

The Direct Insertion Probe (DIP) is widely used to introduce low vapor pressure liquids and
solids into the mass spectrometer. The sample is loaded into a short capillary tube at the end of a
heated sleeve. This sleeve is then inserted through a vacuum lock so the sample is inside the source
region. After the probe is positioned, the temperature of the capillary tube is increased to vaporize the
sample. This probe is used at higher temperatures than are possible with a direct vapor inlet. In
addition, the sample is under vacuum and located close to the source so that lower temperatures are
required for analysis. This is important for analyzing temperature sensitive compounds. Although the
direct insertion probe is more cumbersome than the direct vapor inlet, it is useful for a wider range of
samples.

• DIRECT IONIZATION OF SAMPLE

Unfortunately, some compounds either decompose when heated or have no significant
vapor pressure. These samples may be introduced to the mass spectrometer by direct ionization from
the condensed phase. These direct ionization techniques are used for liquid chromatography/mass
spectrometry, glow discharge mass spectrometry, fast atom bombardment and laser ablation. The
development of new ionization techniques is an active research area and these techniques are rapidly
evolving. Direct ionization is discussed in greater detail in the next sectio

IONISATION SOURCE:
The ion source is the part of the mass spectrometer that ionizes the material under analysis (the
analyte). The ions are then transported by magnetic or electric fields to the mass analyzer. Techniques
for ionization have been key to determining what types of samples can be analyzed by mass
spectrometry. Electron ionization and chemical ionization are used for gases and vapors.

1. CHEMICAL IONIZATIO
Chemical ionization (CI) is an ionization technique used in mass spectrometry. Chemical
ionization is a lower energy process than electron ionization. The lower energy yields less
fragmentation, and usually a simpler spectrum. A typical CI spectrum has an easily identifiable
molecular ion.

Mechanism
In a CI experiment, ions are produced through the collision of the analyte with ions of a
reagent gas that are present in the ion source. Some common reagent gases include: methane,
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ammonia, and isobutane. Inside the ion source, the reagent gas is present in large excess compared to
the analyte. Electrons entering the source will preferentially ionize the reagent gas. The resultant
collisions with other reagent gas molecules will create ionization plasma. Positive and negative ions of
the analyte are formed by reactions with this plasma.

Primary Ion Formation

Secondary Reagent Ions

Product Ion Formation

(Protonation)

(H − abstraction)

(Adduct formation)

(Charge exchange)

Self chemical ionization occurs when the reagent ion is an ionized form of the analyte.

Negative chemical ionization (NCI)

Chemical ionization for gas phase analysis is either positive or negative. Almost all neutral analytes
can form positive ions through the reactions described above.

In order to see a response by negative chemical ionization, the analyte must be capable of producing a
negative ion (stabilize a negative charge) for example by electron capture ionization. Because not all
analytes can do this, using NCI provides a certain degree of selectivity that is not available with other,
more universal ionization techniques (EI, PCI). NCI can be used for the analysis of compounds
containing acidic groups or electronegative elements (especially halogens).

Because of the high electronegativity of halogen atoms, NCI is a common choice for their analysis.
This includes many groups of compounds, such as PCBs, pesticides, and fire retardants. Most of these
compounds are environmental contaminants, thus much of the NCI analysis that takes place is done
under the auspices of environmental analysis.
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2. ATMOSPHERIC PRESSURE CHEMICAL IONIZATION (APCI)

Chemical ionization in an atmospheric pressure electric discharge is called atmospheric pressure

chemical ionization. The analyte is a gas or liquid spray and ionization is accomplished using an
atmospheric pressure corona discharge. This ionization method is often coupled with high performance
liquid chromatography where the mobile phase containing eluting analyte sprayed with high flow rates
of nitrogen and the aerosol spray is subjected to a corona discharge to create ions.

Atmospheric pressure chemical ionization (APCI) is an ionization method used in mass spectrometry.
It is a form of chemical ionization which takes place at atmospheric pressure.

How it works

APCI allows for the high flow rates typical of standard bore HPLC to be used directly, often without
diverting the larger fraction of volume to waste. Typically the mobile phase containing eluting analyte
is heated to relatively high temperatures (above 400 degrees Celsius), sprayed with high flow rates of
nitrogen and the entire aerosol cloud is subjected to a corona discharge that creates ions. Often APCI
can be performed in a modified ESI source. This is basically a gas phase ionisation, unlike ESI which
is a liquid phase ionisation process. Also, we can use nonpolar solvent for solution making instead of
polar solvent for supporting ions in solution as gaseous state conversion of solvent before reaching to
corona discharge pin is carried out here, which well supports the ions formed. Typically, APCI is a less
"soft" ionization technique than ESI, i.e. it generates more fragment ions relative to the parent ion.

Schematic diagram of atmospheric chemical ionisation

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3. ELECTRON IONIZATION

Electron ionization (EI, formerly known as electron impact) is an ionization method in which energetic
electrons interact with gas phase atoms or molecules to produce ions. This technique is widely used in
mass spectrometry, particularly for gases and volatile organic molecules.

Principle of operation

Diagram representing an electron ionization ion source

The following gas phase reaction describes the electron ionization process

where M is the analyte molecule being ionized, e- is the electron and M+• is the resulting ion.

In an EI ion source, electrons are produced through thermionic emission by heating a wire filament
that has electric current running through it. The electrons are accelerated to 70 eV in the region
between the filament and the entrance to the ion source block. The accelerated electrons are then
concentrated into a beam by being attracted to the trap electrode. The sample under investigation
which contains the neutral molecules is introduced to the ion source in a perpendicular direction to the
e- beam. Upon interaction with the e- beam, the analyte molecules ionize to radical cations which are
then directed towards the mass analyzer by a repeller electrode. Due to the high energy electrons and
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the initial thermal distribution of the neutral molecules, the ionization process frequently causes
cleavage reactions that give rise to fragment ions, which can convey structural information about the
analyte.

The ionization efficiency and production of fragment ions depends strongly on the chemistry of the
analyte and the energy of the electrons. At low energies (around 20 eV), the interactions between the
electrons and the analyte molecules do not transfer enough energy to cause ionization. At around 70
eV, the de Broglie wavelength of the electrons matches the length of typical bonds in organic
molecules (about 0.14 nm) and energy transfer to organic analyte molecules is maximized, leading to
the strongest possible ionization and fragmentation. Under these conditions, about 1 in 1000 analyte
molecules in the source are ionized. At higher energies, the de Broglie wavelength of the electrons
becomes smaller than the bond lengths in typical analytes; the molecules then become "transparent" to
the electrons and ionization efficiency decreases.

4. ELECTROSPRAY IONIZATION

Electrospray (nanoSpray) ionization source

Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions. It is

especially useful in producing ions from macromolecules because it overcomes the propensity of these
molecules to fragment when ionized. The development of electrospray ionization for the analysis of
biological macromolecules was rewarded with the attribution of the Nobel Prize in Chemistry to John
Bennett Fenn in 2002.

Mass spectrometry using ESI is called electrospray ionization mass spectrometry (ESI-MS) or, less
commonly, electrospray mass spectrometry (ES-MS)

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Fenn's first electrospray ionization source (top) coupled to a single quadrupole mass spectrometer

IONIZATION MECHANISM

The liquid containing the analyte(s) of interest is dispersed by electrospray into a fine aerosol. Because
the ion formation involves extensive solvent evaporation, the typical solvents for electrospray
ionization are prepared by mixing water with volatile organic compounds (e.g. methanol, acetonitrile).
To decrease the initial droplet size, compounds that increase the conductivity (e.g. acetic acid) are
customary added to the solution. Large-flow electrosprays can benefit from additional nebulization by
an inert gas such as nitrogen. The aerosol is sampled into the first vacuum stage of a mass
spectrometer through a capillary, which can be heated to aid further solvent evaporation from the
charged droplets. The solvent evaporates from a charged droplet until it becomes unstable upon
reaching its Rayleigh limit. At this point, the droplet deforms and emits charged jets in a process
known as Rayleigh fission. During the fission, the droplet loses a small percentage of its mass along
with a relatively large percentage of its charge

There are two major theories that explain the final production of gas-phase ions:

• The Ion Evaporation Model (IEM) suggests that as the droplet reaches a certain radius the
field strength at the surface of the droplet becomes large enough to assist the field desorption of
solvated ions.

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 • The Charged Residue Model (CRM) suggests that electrospray droplets undergo evaporation
and fission cycles, eventually leading progeny droplets that contain on average one analyte ion
or less. The gas-phase ions form after the remaining solvent molecules evaporates, leaving the
analyte with the charges that the droplet carried.

While there is no definite scientific proof, a large body of indirect evidence suggests that small ions are
liberated into the gas phase through the ion evaporation mechanism, while larger ions form by charged
residue mechanism.

The ions observed by mass spectrometry may be quasimolecular ions created by the addition of a
proton (a hydrogen ion) and denoted [M + H]+, or of another cation such as sodium ion, [M + Na]+, or
the removal of a proton, [M − H]−. Multiply-charged ions such as [M + nH]n+ are often observed. For
large macromolecules, there can be many charge states, resulting in a characteristic charge state
envelope. All these are even-electron ion species: electrons (alone) are not added or removed, unlike in
some other ionization sources. The analytes are sometimes involved in electrochemical processes,
leading to shifts of the corresponding peaks in the mass spectrum.

APPLICATIONS

1.Liquid chromatography–mass spectrometry (LC-MS)

Electrospray ionization is the ion source of choice to couple liquid chromatography with mass
spectrometry. The analysis can be performed online, by feeding the liquid eluting from the LC column
directly to an electrospray, or offline, by collecting fractions to be later analyzed in a classical
nanoelectrospray-mass spectrometry setup.

2. Noncovalent gas phase interactions

Electrospray ionization is also ideal in studying noncovalent gas phase interactions. The electrospray
process is capable of transferring liquid-phase noncovalent complexes into the gas phase without
disrupting the noncovalent interaction. This means that a cluster of two molecules can be studied in the
gas phase by other mass spectrometry techniques. An interesting example of this is studying the
interactions between enzymes and drugs which are inhibitors of the enzyme. Because inhibitors
generally work by noncovalently binding to its target enzyme with reasonable affinity the noncovalent
complex can be studied in this way. Competition studies have been done in this way to screen for
potential new drug candidates

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5. MATRIX ASSISTED LASER DESORPTION IONISATION

Matrix Assisted Laser Desorption Ionisation (MALDI) (F. Hillenkamp, M. Karas, R. C. Beavis, B.
T. Chait, Anal. Chem., 1991, 63, 1193) deals well with thermolabile, non-volatile organic compounds
especially those of high molecular mass and is used successfully in biochemical areas for the analysis
of proteins, peptides, glycoproteins, oligosaccharides, and oligonucleotides. It is relatively
straightforward to use and reasonably tolerant to buffers and other additives. The mass accuracy
depends on the type and performance of the analyser of the mass spectrometer, but most modern
instruments should be capable of measuring masses to within 0.01% of the molecular mass of the
sample, at least up to ca. 40,000 Da.

MALDI is based on the bombardment of sample molecules with a laser light to bring about sample
ionisation. The sample is pre-mixed with a highly absorbing matrix compound for the most consistent
and reliable results and a low concentration of sample to matrix work best. The matrix transforms the
laser energy into excitation energy for the sample, which leads to sputtering of analyte and matrix
ions from the surface of the mixture. In this way energy transfer is efficient and also the analyte
molecules are spared excessive direct energy that may otherwise cause decomposition. Most
commercially available MALDI mass spectrometers now have a pulsed nitrogen laser of wavelength
337 nm.

Matrix assisted laser desorption ionisation (MALDI)

The sample to be analysed is dissolved in an appropriate volatile solvent, usually with a trace of
trifluoroacetic acid if positive ionisation is being used, at a concentration of ca. 10 pmol/µL and an
aliquot (1-2 µL) of this removed and mixed with an equal volume of a solution containing a vast
excess of a matrix. A range of compounds is suitable for use as matrices: sinapinic acid is a common
one for protein analysis while alpha-cyano-4-hydroxycinnamic acid is often used for peptide

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analysis. An aliquot (1-2 µL) of the final solution is applied to the sample target which is allowed to
dry prior to insertion into the high vacuum of the mass spectrometer. The laser is fired, the energy
arriving at the sample/matrix surface optimised, and data accumulated until a m/z spectrum of
reasonable intensity has been amassed. The time-of-flight analyser separates ions according to their
mass (m)-to-charge (z) (m/z) ratios by measuring the time it takes for ions to travel through a field
free region known as the flight, or drift, tube. The heavier ions are slower than the lighter ones.

The m/z scale of the mass spectrometer is calibrated with a known sample that can either be analysed
independently (external calibration) or pre-mixed with the sample and matrix (internal calibration).

Simplified schematic of MALDI-TOF mass spectrometry (linear mode)

Sample target for a Maldi Tof Spectrometer

MALDI is also a "soft" ionisation method and so results predominantly in the generation of singly
charged molecular-related ions regardless of the molecular mass, hence the spectra are relatively
easy to interpret. Fragmentation of the sample ions does not usually occur.
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In positive ionisation mode the protonated molecular ions (M+H+) are usually the dominant species,
although they can be accompanied by salt adducts, a trace of the doubly charged molecular ion at
approximately half the m/z value, and/or a trace of a dimeric species at approximately twice the m/z
value. Positive ionisation is used in general for protein and peptide analyses.

In negative ionisation mode the deprotonated molecular ions (M-H-) are usually the most abundant
species, accompanied by some salt adducts and possibly traces of dimeric or doubly charged materials.
Negative ionisation can be used for the analysis of oligonucleotides and oligosaccharides.

Positive ionisation MALDI m/z spectrum of a peptide mixture using alpha-cyano-4-

hydroxycinnamic acid as matrix

USES:-

In Biochemistry

In proteomics, MALDI is used for the identification of proteins isolated through gel electrophoresis:
SDS-PAGE, size exclusion chromatography, and two-dimensional gel electrophoresis. One method
used is peptide mass fingerprinting by MALDI-MS, or with post ionisation decay or collision-induced
dissociation (further use see mass spectrometry).

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In Organic Chemistry

Some synthetic macromolecules, such as catenanes and rotaxanes, dendrimers and hyperbranched
polymers, and other assemblies, have molecular weights extending into the thousands or tens of
thousands, where most ionization techniques have difficulty producing molecular ions. MALDI is a
simple and rapid analytical method that can allow chemists to analyze the results of such syntheses and
verify their results.

In polymer chemistry

In polymer chemistry MALDI can be used to determine the molar mass distribution. Polymers with
polydispersity greater than 1.2 are difficult to characterize with MALDI due to the signal intensity
discrimination against higher mass oligomers.

Reproducibility and performance

The sample preparation for MALDI is important for the result. Inorganic salts which are also part of
protein extracts interfere with the ionization process. The salts are removed by solid phase extraction
or washing the final target spots with water. Both methods can also remove other substances from the
sample. The matrix protein mixture is not homogenous because the polarity difference leads to a
separation of the two substances during crystallization. The spot diameter of the target is much larger
than that of the laser, which makes it necessary to do several laser shots at different places of the
target, to get the statistical average of the substance concentration within the target spot. The matrix
composition, the addition of trifluoroacetic acid and formic acid, delay between laser pulses, delay
time of the acceleration power, laser wavelength, energy density of the laser and the impact angle of
the laser on the target are among others the critical values for the quality and reproducibility of the
method

6. FIELD DESORPTION IONISATION:

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Schematic of field desorption ionization with emitter at left and mass spectrometer at right

Field desorption (FD)/field ionization (FI) refers to an ion source for mass spectrometry first reported
by Beckey in 1969. In field ionization, a high-potential electric field is applied to an emitter with a
sharp surface, such as a razor blade, or more commonly, a filament from which tiny "whiskers" have
formed. This results in a very high electric field which can result in ionization of gaseous molecules of
the analyte. Mass spectra produced by FI have little or no fragmentation. They are dominated by

molecular radical cations M+. And less often, protonated molecules .

Mechanism

In FD, the analyte is applied as a thin film directly to the emitter, or small crystals of solid materials
are placed onto the emitter. Slow heating of the emitter then begins, by passing a high current through
the emitter, which is maintained at a high potential (e.g. 5 kilovolts). As heating of the emitter
continues low-vapor-pressure materials get desorbed and ionized by alkali metal cation attachment.

Applications

Many earlier applications of FD/FI to analysis of polar and nonvolatile analytes such as polymers and
biological molecules have largely been supplanted by newer ionization techniques. However, FD/FI
remains one of the only ionization techniques that can produce simple mass spectra with molecular
information from hydrocarbons and other particular analytes. The most commonly encountered
application of FD/FI at the present time is the analysis of complex mixtures of hydrocarbons such as
that found in petroleum fractions.

Liquid injection

The recently developed liquid injection FD ionization (LIFDI) technique "presents a major
breakthrough for FD-MS of reactive analytes" : Transition metal complexes are neutral and due to
their reactivity, do not undergo protonation or ion attachment. They benefit from both: the soft FD
ionization and the safe and simple LIFDI transfer of air/moisture sensitive analyte solution. This
transfer occurs from the Schlenk flask to the FD emitter in the ion source through a fused silica
capillary without breaking the vacuum.

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7. FAST ATOM BOMBARDMENT IONISATION:

Fast atom bombardment (FAB) is an ionization technique used in mass spectrometry. The material to
be analyzed is mixed with a non-volatile chemical protection environment called a matrix and is
bombarded under vacuum with a high energy (4000 to 10,000 electron volts) beam of atoms. The
atoms are typically from an inert gas such as argon or xenon. Common matrices include glycerol,
thioglycerol, 3-nitrobenzyl alcohol (3-NBA), 18-Crown-6 ether, 2-nitrophenyloctyl ether, sulfolane,
diethanolamine, and triethanolamine. This technique is similar to secondary ion mass spectrometry and
plasma desorption mass spectrometry.

How it works

FAB is a relatively soft ionization technique and produces primarily intact protonated molecules
denoted as [M+H]+ and deprotonated molecules such as [M-H]-. The nature of its ionization products
places it close to electrospray and MALDI

The first example of the practical application of this technique was the elucidation of the amino acid
sequence of the oligopeptide efrapeptin D. This contained a variety of very unusual amino acid
residues.[6] The sequence was shown to be: N-acetyl-L-pip-AIB-L-pip-AIB-AIB-L-leu-beta-ala-gly-
AIB-AIB-L-pip-AIB-gly-L-leu-L-iva-AIB-X. PIP = pipecolic acid, AIB = alpha-amino-isobutyric
acid,leu = leucine, iva = isovaline, gly = glycine. This is a potent inhibitor of the mitochodrial ATPase
activity.

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8. THERMOSPRAY IONISATION:

Thermospray is a form of atmospheric pressure ionization in mass spectrometry. It transfers ions

from the liquid phase to the gas phase for analysis. It is particularly useful in liquid chromatography-
mass spectrometry.

Ionization mechanism

TSP ionization is achieved by passing a pressurized solution through a heated tube which partially
vaporizes the effluent to generate a spray prior to entering the ion source. Droplets from the spray
contain a statistical imbalance of charges originating from charged solutes present in the solution. The
droplets gradually decrease in size by evaporation of neutral solvent molecules until the droplet
reaches a size at which the charge repulsion forces overcome the cohesive forces of the droplet.

ANALYZERS

Mass analyzer technologies

Mass analyzers separate the ions according to their mass-to-charge ratio. The following two laws
govern the dynamics of charged particles in electric and magnetic fields in vacuum:

1.Lorentz force law

2.Newton's second law of motion in non-relativistic case, i.e. valid only at ion velocity much lower
than the speed of light)

Here F is the force applied to the ion, m is the mass of the ion, a is the acceleration, Q is the ion
charge, E is the electric field, and v x B is the vector cross product of the ion velocity and the magnetic
field

Equating the above expressions for the force applied to the ion yields:

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This differential equation is the classic equation of motion for charged particles. Together with the
particle's initial conditions, it completely determines the particle's motion in space and time in terms of
m/Q. Thus mass spectrometers could be thought of as "mass-to-charge spectrometers". When
presenting data, it is common to use the (officially) dimensionless m/z, where z is the number of
elementary charges (e) on the ion (z=Q/e). This quantity, although it is informally called the mass-to-
charge ratio, more accurately speaking represents the ratio of the mass number and the charge number,
z.

There are many types of mass analyzers, using either static or dynamic fields, and magnetic or electric
fields, but all operate according to the above differential equation. Each analyzer type has its strengths
and weaknesses. Many mass spectrometers use two or more mass analyzers for tandem mass
spectrometry (MS/MS). In addition to the more common mass analyzers listed below, there are others
designed for special situations.

1. QUADRUPOLE MASS ANALYZER

The quadrupole mass analyzer is one type of mass analyzer used in mass spectrometry. As the name
implies, it consists of 4 circular rods, set perfectly parallel to each other. In a quadrupole mass
spectrometer the quadrupole mass analyzer is the component of the instrument responsible for
filtering sample ions, based on their mass-to-charge ratio (m/z). Ions are separated in a quadrupole
based on the stability of their trajectories in the oscillating electric fields that are applied to the rods.

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How it works

Image from "Apparatus For Separating Charged Particles Of Different Specific Charges" Patent
number: 2939952

The quadrupole consists of four parallel metal rods. Each opposing rod pair is connected together
electrically and a radio frequency voltage is applied between one pair of rods and the other. A direct
current voltage is then superimposed on the R.F. voltage. Ions travel down the quadrupole between the
rods. Only ions of a certain 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 or allows the operator to scan for a range of m/z-values by continuously varying the applied
voltage.

Applications

These mass spectrometers excel at applications where particular ions of interest are being studied
because they can stay tuned on a single ion for extended periods of time. One place where this is useful

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is in liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry where they
serve as exceptionally high specificity detectors. Quadrupole instruments are often reasonably priced
and make good multi-purpose instruments.

TRIPLE QUADRUPOLES

A linear series of three quadrupoles can be used; known as a triple quadrupole mass spectrometer. The
first (Q1) and third (Q3) quadrupoles act as mass filters, and the middle (q2) quadrupole is employed as
a collision cell. This collision cell is an RF only quadrupole (non-mass filtering) using Ar, He or N gas
(~10-3 Torr, ~30 eV) to induce collision induced dissociation of selected parent ion(s) from Q 1.
Subsequent fragments are passed through to Q3 where they may be filtered or scanned fully.

This process allows for the study of fragments (daughter ions) which are crucial in structural
elucidation. For example, the Q1 may be set to "filter" for a drug ion of a known mass, which is
fragmented in q2. The third quadrupole (Q3) can then be set to scan the entire m/z range, giving
information on the sizes of the fragments made. Thus, the structure of the original ion can be deduced.

The arrangement of three quadrupoles was first developed by Jim Morrison of LaTrobe University,
Australia for the purpose of studying the photodissociation of gas-phase ions. The first triple-
quadrupole mass spectrometer was developed at Michigan State University by Dr. Christie Enke and
graduate student Richard Yost in the late 1970's.

2. TIME-OF-FLIGHT (TOF) MASS ANALYSIS

Theory and History:

Time-of-flight mass spectrometry (TOF-MS) is probably the simplest method of mass measurement
to conceptualise, although there are hidden complexities when it comes to higher resolution
applications. The first commercial TOF instrument was marketed by the Bendix Corporation in the late
1950's. Their design was based on the Wiley & MacLaren instrument that was published in 1955 [1].
TOF-MS has really come into its own in recent years as being an essential instrument for biological
analysis applications - this is especially the case with the coupling of TOF-MS to MALDI and ESI
ionisation methods and the development of high-resolution and hybrid instruments (for example Q-
TOF and TOF-TOF configurations). The inherent characteristics of TOF-MS are extreme sensitivity
(all ions are detected), almost unlimited mass range and speed of analysis (modern instruments can
obtain full spectra in seconds). This makes TOF-MS one of the most desirable methods of mass
analysis.

Fig. 1: A Schematic of a Time-of-Flight mass spectrometer operating in Reflectron Mode

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 The general set up of TOF is shown in Fig. 1. The ions are introduced either directly from the source
of the instrument or from a previous analyser (in the case of Q-TOF) as a pulse. This results in all the
ions receiving the same initial kinetic energy. As they then pass along the field free drift zone, they are
separated by their masses, lighter ions travel faster. This enables the instrument to record all ions as
they arrive at the detector and so accounts for the techniques high sensitivity. The equation governing
TOF separation is:

m/z is mass-to-charge ratio of the ion

E is the extraction pulse potential
s is the length of flight tube over which E is applied
d is the length of field free drift zone
t is the measured time-of-flight of the ion

Theoretically then, all the ions are given the same initial kinetic energy by the extraction pulse and
then drift along the field free drift zone where they will be separated so that all ions of the same m/z
arrive at the detector at the same time. In practice, the pulse is not felt by all ions to the same intensity
and so a kinetic energy distribution for each discrete m/z exists. This lowers the resolution by creating
a time-of-flight distribution for each m/z [2]. This is relatively easily corrected for by the application of
a reflectron at the end of the drift zone [3]. This consists of a series of electric fields which repulse the
ions back along the flight tube - usually at a slightly displaced angle (see figure) - resulting in a
refocusing of ions with the same m/z value on the reflectron detector.

22
 ION TRAP ANALYSER:
3.

An ion trap is a combination of electric or magnetic fields that captures ions in a region of a vacuum
system or tube. Ion traps have a number of scientific uses such as trapping ions while the ion's
quantum state is manipulated and mass spectrometery. The two most common types of ion traps are
the Penning trap and the Paul trap (quadrupole ion trap).

When using ion traps for scientific studies of quantum state manipulation, the Paul trap is most often
used. This work may lead to a trapped ion quantum computer and has already been used to create the
world’s most accurate atomic clocks.

An ion trap mass spectrometer may incorporate a Penning trap (Fourier transform ion cyclotron
resonance), Paul trap or the Kingdon trap. The Orbitrap, introduced in 2005, is based on the Kingdon
trap. Other types of mass spectrometers may also use a linear quadrupole ion trap as a selective mass
filter.

In an electron gun (a device emitting high-speed electrons, such as those in CRTs), an ion trap may be
implemented above the cathode (using an extra, positively-charged electrode between the cathode and

23
the extraction electrode) to prevent its degradation by positive ions accelerated backward by the fields
intended to pull electrons away from the cathode.

4. FOURIER TRANSFORM ION CYCLOTRON RESONANCE

ANALYZER:

Fourier transform mass spectrometry, or more precisely Fourier transform ion cyclotron resonance
MS, measures mass by detecting the image current produced by ions cyclotroning in the presence of a
magnetic field. Instead of measuring the deflection of ions with a detector such as an electron
multiplier, the ions are injected into a Penning trap (a static electric/magnetic ion trap) where they
effectively form part of a circuit. Detectors at fixed positions in space measure the electrical signal of
ions which pass near them over time, producing a periodic signal. Since the frequency of an ion's
cycling is determined by its mass to charge ratio, this can be deconvoluted by performing a Fourier
transform on the signal. FTMS has the advantage of high sensitivity (since each ion is "counted" more
than once) and much higher resolution and thus precision

Ion cyclotron resonance (ICR) is an older mass analysis technique similar to FTMS except that ions
are detected with a traditional detector. Ions trapped in a Penning trap are excited by an RF electric
field until they impact the wall of the trap, where the detector is located. Ions of different mass are
resolved according to impact time.

24
 5. ORBITRAP ANALYZER:

An orbitrap is a type of mass spectrometer invented by Alexander Makarov. It consists of an outer

barrel-like electrode and a coaxial inner spindle-like electrode that form an electrostatic field with
quadro-logarithmic potential distribution.

PRINCIPLE OF OPERATION

Figure 3 from U.S. patent 6995364 indicating the orbitrap (130), central electrode (140), two outer
electrodes (160 and 170), amplifier (180) and field compensator (200).

In an orbitrap, ions are injected tangentially into the electric field between the electrodes and trapped
because their electrostatic attraction to the inner electrode is balanced by centrifugal forces. Thus, ions
cycle around the central electrode in rings. In addition, the ions also move back and forth along the
axis of the central electrode. Therefore, ions of a specific mass-to-charge ratio move in rings which
oscillate along the central spindle. The frequency of these harmonic oscillations is independent of the
ion velocity and is inversely proportional to the square root of the mass-to-charge ratio (m/z or m/q).
By sensing the ion oscillation similar as in the FTICR-MS, the trap can be used as a mass analyzer.
Orbitraps have a high mass accuracy (1–2 ppm), a high resolving power (up to 200,000) and a high
dynamic range (around 5000).

25
Ion trajectories in an Orbitrap mass spectrometer.

Like FTICR-MS the orbitrap resolving power is proportional to the number of harmonic oscillations of
the ions, as a result the resolving power is inversely proportional to the square root of m/z and
proportional to acquisition time. Scigelova and Makarov show the resolving power to be 7500 per 0.1
second transient at m/z 400. A transient being the duration that the time domain signal is acquired for.
The resolving power decreases further as the m/z value increases so that at 4 times the m/z value (1600)
the resolving power has halved to 3750 per 0.1 second. Approximately 0.1 seconds per transient is
required for data processing, thus a 0.1 second transient has a cycle time of 0.2 seconds. These effects
in combination result in a resolving power of 7500 at m/z 400 and 3750 at m/z 1600 when the orbitrap
is operating at 5 acquisitions per second.

The linear trap quadrupole (LTQ) can be used as a front end for the orbitrap.

DETECTOR

A continuous dynode particle multiplier detector.

The final element of the mass spectrometer is the detector. The detector records either the charge
induced or the current produced when an ion passes by or hits a surface. In a scanning instrument, the
signal produced in the detector during the course of the scan versus where the instrument is in the scan
(at what m/Q) will produce a mass spectrum, a record of ions as a function of m/Q

26
 1) ELECTRON MULTIPLIER DETECTOR:
Continuous dynode electron multiplier

An electron multiplier (continuous dynode electron multiplier) is a vacuum-tube structure that

multiplies incident charges. In a process called secondary emission, a single electron can, when
bombarded on metal (or PbO coated surface) induce emission of roughly 1 to 3 electrons. If an electric
potential is applied between this metal plate and yet another, the emitted electrons will accelerate to
the next metal plate and induce secondary emission of still more electrons. This can be repeated a
number of times, resulting in a large shower of electrons all collected by a metal anode, all having
been triggered by just one.

Operation

The avalanche can be triggered by any charged particle hitting the starting electrode with sufficient
energy to cause secondary emission. Hence the electron multiplier is often used as an ion detector. It
could also be triggered by a photon causing vacuum photoemission of at least one electron. In a
photomultiplier tube, a photo-emissive surface is followed by an electron multiplier with several
sequential multiplying electrodes called dynodes. Because these electrodes are separate from each
other, this might be called a "discrete-dynode" multiplier. A voltage divider chain of resistors is
usually used to place each dynode at a potential 100-200v more positive than the previous one.

A "continuous-dynode" structure is feasible if the material of the electrodes has a high resistance, so
that the functions of secondary-emission and voltage-division are merged. This is often built as a
funnel of glass coated inside with a thin film of semi-conducting material, with negative high voltage
applied at the wider input end, and positive voltage near ground applied at the narrower output end.
Electrons emitted at any point are accelerated a modest distance down the funnel before impacting the
surface, perhaps on the opposite side of the funnel. At the destination end a separate electrode (anode)
remains necessary to collect the multiplied electrons. This structure is also known as (single) channel
electron multiplier (CEM), and one of the most common is sold under the trade name Channeltron.

Geometry of continuous-dynode electron multiplier is called the microchannel plate. It may be

considered a 2-dimensional parallel array of very small continuous-dynode electron multipliers, built
together and powered in parallel too. Each microchannel is generally parallel-walled, not tapered or
funnel-like

27
 2) FARADAY CUP DETECTOR:

A Faraday cup is a metal (conductive) cup designed to catch charged particles in vacuum. The
resulting current can be measured and used to determine the number of ions or electrons hitting the
cup.[1] The Faraday cup is named after Michael Faraday who first theorized ions around 1830.

How it works

When a beam or packet of Ions hits the metal it gains a small net charge while the ions are neutralized.
The metal can then be discharged to measure a small current equivalent to the number of impinging
ions. Essentially the faraday cup is part of a circuit where ions are the charge carriers in vacuum and
the faraday cup is the interface to the solid metal where electrons act as the charge carriers (as in most
circuits). By measuring the electrical current (the number of electrons flowing through the circuit per
second) in the metal part of the circuit the number of charges being carried by the ions in the vacuum
part of the circuit can be determined. For a continuous ion beam of ions (each with a single charge)

Faraday cup with an electron-supressor plate in front

28
Where N is the number of ions observed in a time t (in seconds), I is the measured current (in amperes)
and e is the elementary charge (about 1.60 × 10-19 C). Thus, a measured current of one nanoamp (10-9
A) corresponds to about 6 billion ions striking the faraday cup each second.

Similarly, a Faraday cup can act as a collector for electrons in a vacuum (for instance from an electron
beam). In this case electrons simply hit the metal plate/cup and a current is produced. Faraday cups are
not as sensitive as electron multiplier detectors, but are highly regarded for accuracy because of the
direct relation between the measured current and number of ions.

3. ARRAY DETECTORS
Array detectors can attain very high sensitivities by collecting all the ions of a given mass range
continuously.This contrasts with conventional scanning methods using point detectors (e.g. electron
multipliers) that collect only a small fraction of the ions in each mass channel as the spectrum is
scanned.
Photodiode arrays and similar devices were limited by their low resolving powers and narrow mass
ranges. However, recent improvements have extended the mass range by sequentially stepping
‘scanning arrays’ under computer control. Alternatively, specially designed ion-optical systems have
been developed for use with wide-angle array detectors. Both developments allow reasonable scan
rates for the total spectrum. Array detection can increase the apparent sensitivity by two orders of
magnitude.

THE VACUUM SYSTEM:

All mass spectrometers operate at very low pressure (high vacuum). This reduces the chance of ions
colliding with other molecules in the mass analyzer. Any collision can cause the ions to react,
neutralize, scatter, or fragment. All these processes will interfere with the mass spectrum. To minimize
collisions, experiments are conducted under high vacuum conditions, typically 10-2 to 10-5 Pa (10-4 to
10-7 torr) depending upon the geometry of the instrument. This high vacuum requires two pumping
stages. The first stage is a mechanical pump that provides rough vacuum down to 0.1 Pa (10-3 torr).
The second stage uses diffusion pumps or turbomolecular pumps to provide high vacuum. ICR
instruments have even higher vacuum requirements and often include a cryogenic pump for a third
pumping stage. The pumpingsystem is an important part of any mass spectrometer but a detailed
discussion is beyond thescope of this paper

29
RESOLUTION IN MASS SPECTROMETER
In mass spectrometry, resolution is a measure of the ability to distinguish between two peaks of
different mass-to-charge ratio, m/z, in a mass spectrum. There are several ways to define resolution in
mass spectrometry, therefore it is important to report the method used to determine mass resolution
when reporting its value.

Wher m and delta m are mass numbers of two neighbouring peaks

VALLEY DEFINITION: The valley definition defines Δm as the closest spacing of two peaks
of equal intensity with the valley (lowest value of signal) between them less than aspecifiedfraction of
the peak height. Typical values are 10% or 50%. The value obtained from a 5% peak width is roughly
equivalent to a 10% valley.

DATA AND ANALYSIS

Mass spectrum of a peptide showing the isotopic distribution

DATA REPRESENTATIONS

Mass spectrometry produces various types of data. The most common data representation is the mass
spectrum certain types of mass spectrometry data are best represented as a mass chromatogram. Types
of chromatograms include selected ion monitoring (SIM), total ion current (TIC), and selected reaction
monitoring chromatogram (SRM), among many others. Other types of mass spectrometry data are well
represented as a three dimensional contour map. In this form, the mass-to-charge, m/z is on the x-axis,
intensity the y-axis, and an additional experimental parameter, such as time, is recorded on the z-axis.

30
 DATA ANALYSIS

Basics

Mass spectrometry data analysis is a complicated subject matter that is very specific to the type of
experiment producing the data. There are general subdivisions of data that are fundamental to
understand any data.

Many mass spectrometers work in either negative ion mode or positive ion mode. It is very important
to know whether the observed ions are negatively or positively charged. This is often important in
determining the neutral mass but it also indicates something about the nature of the molecules.

Different types of ion source result in different arrays of fragments produced from the original
molecules. An electron ionization source produces many fragments and mostly odd electron species
with one charge; whereas an electrospray source usually produces quasimolecular even electron
species that may be multiply charged. Tandem mass spectrometry purposely produces fragment ions
post-source and can drastically change the sort of data achieved by an experiment.

By understanding the origin of a sample, certain expectations can be assumed as to the component
molecules of the sample and their fragmentations. A sample from a synthesis/manufacturing process
will probably contain impurities chemically related to the target component. A relatively crudely
prepared biological sample will probably contain a certain amount of salt, which may form adducts
with the analyte molecules in certain analyses.

Results can also depend heavily on how the sample was prepared and how it was run/introduced. An
important example is the issue of which matrix is used for MALDI spotting, since much of the
energetics of the desorption/ionization event is controlled by the matrix rather than the laser power.
Sometimes samples are spiked with sodium or another ion-carrying species to produce adducts rather
than a protonated species.

The greatest source of trouble when non-mass spectrometrists try to conduct mass spectrometry on
their own or collaborate with a mass spectrometrist is inadequate definition of the research goal of the
experiment. Adequate definition of the experimental goal is a prerequisite for collecting the proper
data and successfully interpreting it. Among the determinations that can be achieved with mass
spectrometry are molecular mass, molecular structure, and sample purity. Each of these questions
requires a different experimental procedure. Simply asking for a "mass spec" will most likely not
answer the real question at hand.

31
INTERPRETATION OF MASS SPECTRA

Since the precise structure or peptide sequence of a molecule is deciphered through the set of fragment
masses, the interpretation of mass spectra requires combined use of various techniques. Usually the
first strategy for identifying an unknown compound is to compare its experimental mass spectrum
against a library of mass spectra. If the search comes up empty, then manual interpretation or software
assisted interpretation of mass spectra are performed. Computer simulation of ionization and
fragmentation processes occurring in mass spectrometer is the primary tool for assigning structure or
peptide sequence to a molecule. An a priori structural information is fragmented in silico and the
resulting pattern is compared with observed spectrum. Such simulation is often supported by a
fragmentation library that contains published patterns of known decomposition reactions. Software
taking advantage of this idea has been developed for both small molecules and proteins.

Another way of interpreting mass spectra involves spectra with accurate mass. A mass-to-charge ratio
value (m/z) with only integer precision can represent an immense number of theoretically possible ion
structures. More precise mass figures significantly reduce the number of candidate molecular formulas,
albeit each can still represent large number of structurally diverse compounds. A computer algorithm
called formula generator calculates all molecular formulas that theoretically fit a given mass with
specified tolerance.

A recent technique for structure elucidation in mass spectrometry, called precursor ion fingerprinting
identifies individual pieces of structural information by conducting a search of the tandem spectra of
the molecule under investigation against a library of the product-ion spectra of structurally
characterized

MASS SPECTRUM ANALYSIS

32
Mass spectrum analysis is an integral part of spectroscopy and mass spectrometry dealing with the
interpretation of mass spectra Organic chemists obtain mass spectra of chemical compounds as part of
structure elucidation and the analysis is part of every organic chemistry curriculum.

BASIC PEAKS

Mass spectra have several distinct sets of peaks or ions:

1. the molecular ion,

2. isotope peaks
3. fragmentation peaks
4. metastable peaks
5. rearrangement ion
6. Multiply charged ion
7. Negative ion

1. the molecular ion

Mass spectra first of all display the molecular ion (or parent ion) peak which is a radical cation M+. as
a result of removing one electron from the molecule. In the spectrum for toluene for example the
molecular ion peak is located at 92 m/e corresponding to its molecular mass. The molecular ion peak
does not always appear or can be weak. The height of the molecular ion peak diminishes with
branching and with increasing mass in a homologous series. Identifying the molecular ion can be
difficult. A useful aid is the nitrogen rule: if the mass is an even number, the compound contains no
nitrogen or an even number of nitrogen’s. Molecular ion peaks are also often preceded by a M-1 or M-
2 peak resulting from loss of a hydrogen radical or dihydrogen.

2. Isotope ion

More peaks are visible with m/e ratios larger than the molecular ion peak due to isotope distributions.
The value of 92 in the toluene example corresponds to the monoisotopic mass of a molecule of toluene
entirely composed of the most abundant isotopes (1H and 12C). The so-called M+1 peak corresponds to
a fraction of the molecules with one higher isotope incorporated ((2H or 13C) and the M+2 peak has two
higher isotopes. The natural abundance of the higher isotopes is low for frequently encountered
elements such as hydrogen, carbon and nitrogen and the intensity of isotope peaks subsequently low
and the intensity quickly diminishes with total mass. In halogens on the other hand higher isotopes
have a large abundance which results in a specific mass signature for halogen containing compounds.

33
 3. Fragmentation ion

Peaks with mass less that the molecular ion are the result of fragmentation of the molecule. These
peaks are called daughter peaks. The peak with the highest ratio is called the base peak which is not
necessary the molecular ion. Many reaction pathways exist for fragmentation but only newly formed
cations will show up in the mass spectrum and not radical fragments or neutral fragments.

4. The metastable ion

Metastable peaks are broad peaks at non-integer mass values. These peaks result from molecular
fragments with lower kinetic energy because of fragmentations taking place ahead of the ionization
chamber. They are not of analytical value.

5. Rearrangement ion

Fragment ions formed by the intramolecular rearrangements involving migration of hydrogen atoms
from one part of the ion to other part are called rearrangement ions Rearrangement process are
common in unsaturated compounds and are fovoured even at low electron voltage

The most common example is “McLafferty Rearrangement”

6. Multiply charged ions

In mass spectrometer, the ions are carrying a single positive charge. How ever, sometime doubly or
even triply charged ions are found in mass spectrum. The doubly or triply charged ions are recorded at
a half or a third of the m/e value of the singly charged ions. The formation of these multiply charged
ions are more common in hetero atomic molecules

7. Negative ions: Though in ionisation process positive ions are produced, in few cases negative
ions do get formed these result due to the capture of electron by a molecule during collision of
molecules. Their formation is very rare but these can be produced in three ways

34
1 dissociatin resonance capture

2. Resonance capture

3. Ion pair production

The negative ions are focussed by reversing the field in the mass spectrometer.these are not very useful
in structural determinations

Ex: o-, oh-.c2h-

FRAGMENTATION

The fragmentation pattern not only allows the determination of the mass of an unknown compound but
also allows guessing the molecular structure especially in combination the calculation of the degree of
unsaturation from the molecular formula (when available). Neutral fragments frequently lost are
carbon monoxide, ethylene, water, ammonia, and hydrogen sulfide.

Fragmentations arise from:

• Homolysis processes. An example is the cleavage of carbon-carbon bonds next to a heteroatom

In this depiction single-electron movements are indicated by a fishhook arrow.

• Rearrangement reactions, for example a retro Diels-Alder reaction extruding neutral ethylene:

Or the McLafferty rearrangement. As it is not always obvious where a lone electron resides in a
radical cation a square bracket notation is often used.

35
MCLAFFERTY REARRANGEMENT

The McLafferty rearrangement is a reaction observed in mass spectrometry. It is sometimes found

that a molecule containing a keto-group undergoes β-cleavage, with the gain of the γ-hydrogen atom.
This rearrangement may take place by a radical or ionic mechanism.

The reaction

A description of the reaction was first published by the American chemist Fred McLafferty in 1959

ALPHA CLEAVAGE

Alpha cleavage, (α-cleavage) in organic chemistry, refers to the act of breaking the carbon-carbon
bond, [1] adjacent to the carbon bearing a specified functional group.

Generally this topic is discussed when covering mass spectrometry and occurs generally by the same
mechanisms.

For example of a mechanism of alpha cleavage, an electron is knocked off an atom (usually by
electron collision) to form a radical cation. Electron removal generally happens in the following order:
1) lone pair electrons, 2) pi bond electrons, 3) sigma bond electrons

One of the lone pair electrons moves down to form a pi bond with an electron from an adjacent (alpha)
bond.The other electron from the bond moves to an adjacent atom (not one adjacent to the lone pair
atom) creating a radical. This creates a double bond adjacent to the lone pair atom (oxygen is a good
example) and breaks/cleaves the bond from which the two electrons were removed.

Example C-C-(O::)-H > C-C-(O:.+)-H > C' + (C=O:+)-H where: is a lone pair + is a positive charge
and ' is a radical/free electron

In molecules containing carbonyl groups, often competes with McLafferty rearrangement.

36
SOME GENERAL RULES:

• Cleavage occurs at alkyl substituted carbons reflecting the order generally observed in
carbocations.
• Double bonds and arene fragments tend to resist fragmentation.
• Allylic cations are stable and resist fragmentation.
• the even-electron rule stipulates that even-electron species (cations but not radical ions) will
not fragment into two odd-electron species but rather to another cation and a neutral molecule

NITROGEN RULE

The nitrogen rule states that organic compounds containing exclusively hydrogen, carbon, nitrogen,
oxygen, silicon, phosphorus, sulfur, and the halogens either have 1) an odd nominal mass that indicates
an odd number of nitrogen atoms are present or 2) an even nominal mass that indicates an even
number of nitrogen atoms are present in the molecular ion The nitrogen rule is not a rule, per se, as
much as a general principle which may prove useful when attempting to solve organic mass
spectrometry structures.

Formulation of the rule

This rule is derived from the fact that, perhaps coincidentally, for the most common chemical elements
in neutral organic compounds (hydrogen, carbon, nitrogen, oxygen, silicon, phosphorus, sulfur, and the
halogens), elements with even numbered nominal masses form even numbers of covalent bonds, while
elements with odd numbered nominal masses form odd numbers of covalent bonds, with the exception
of nitrogen, which has a nominal (or integer) mass of 14, but has a valency of 3.

It should be noted that the nitrogen rule is only true for neutral structures in which all of the atoms in
the molecule have a number of covalent bonds equal to their standard valency (counting each sigma
bond and pi bond as a separate covalent bond for the purposes of the calculation). Therefore, the rule is
typically only applied to the molecular ion signal in the mass spectrum.

Mass spectrometry generally operates by measuring the mass of ions. If the measured ion is generated
by creating or breaking a single covalent bond (such as protonating an amine to form an ammonium
center or removing a hydride from a molecule to leave a positively charged ion) then the nitrogen rule
becomes reversed (odd numbered masses indicate even numbers of nitrogens and vice versa).
However, for each consecutive covalent bond that is broken or formed, the nitrogen rule again
reverses.

37
Therefore, a more rigorous definition of the nitrogen rule for organic compounds containing
exclusively hydrogen, carbon, nitrogen, oxygen, silicon, phosphorus, sulfur, and the halogens would
be as follows:

An even nominal mass indicates that a net even number of covalent bonds have been broken or formed
and an even number of nitrogen atoms are present, or that a net odd number of covalent bonds have
been broken or formed and an odd number of nitrogen atoms are present. An odd nominal mass
indicates that a net even number of covalent bonds have been broken or formed and an odd number of
nitrogen atoms are present, or that a net odd number of covalent bonds have been broken or formed
and an even number of nitrogen atoms are present.

Inorganic molecules do not necessarily follow the rule. For example the nitrogen oxides NO and NO2
have an odd number of nitrogens but even masses of 30 and 46

ISOTOPE EFFECTS

Isotope peaks within a spectrum can help in structure elucidation. Compounds containing halogens
(especially chlorine and bromine) produce very distinct isotope peaks. The mass spectrum of
methylbromide has two prominent peaks of equal intensity at 94 (M) and 96 (M+2) and then two more
at 79 and 81 belonging to the bromine fragment.

Even when compounds only contain elements with less intense isotope peaks (carbon or oxygen), the
distribution of these peaks can be used to assign the spectrum to the correct compound. For example,
two compounds with identical mass of 150, C8H12N3+ and C9H10O2+, will have two different M+2
intensities which make it possible to distinguish between them.

38
 TOLUENE EXAMPLE

The mass spectrum for toluene has around 30 signals. Several peaks can be rationalized in this
fragmentation pattern.

39
 Natural abundance of some elements

The next table gives the isotope distributions for some elements. Some elements like phosphorus and
fluorine only exist as a single isotope, with a natural abundance of 100%.

Natural abundance of some elements [3]

Isotope % nat. abundance atomic mass isotope % nat. abundance atomic mass
1 12
H 99.985 1.007825 C 98.89 12 (definition)
2 13
H 0.015 2.0140 C 1.11 13.00335
16
O 99.76 15.99491 14N 99.64 14.00307
17
O 0.04 15N 0.36 15.00011
18
O 0.2
28 32
Si 92.23 27.97693 S 95.0 31.97207
29 33
Si 4.67 28.97649 S 0.76 32.97146
30 34
Si 3.10 29.97376 S 4.22 33.96786
35 79
Cl 75.77 34.96885 Br 50.69 78.9183
37 81
Cl 24.23 Br 49.31 80.9163

CHROMATOGRAPHIC TECHNIQUES COMBINED WITH MASS

SPECTROMETRY
• Gas chromatography

40
 • Liquid chromatography
• Ion mobility

1. GAS CHROMATOGRAPHY MASS SPECTROMETRY (GC/MS)

Gas chromatography mass spectrometry (GC/MS) is an instrumental technique, comprising a gas

chromatograph (GC) coupled to a mass spectrometer (MS), by which complex mixtures of chemicals
may be separated, identfied and quantified. This makes it ideal for the analysis of the hundreds of
relatively low molecular weight compounds found in environmental materials. In order for a
compound to be analysed by GC/MS it must be sufficiently volatile and thermally stable. In addition,
functionalised compounds may require chemical modification (derivatization), prior to analysis, to
eliminate undesirable adsorption effects that would otherwise affect the quality of the data obtained.
Samples are usually analyzed as organic solutions consequently materials of interest (e.g. soils,
sediments, tissues etc.) need to be solvent extracted and the extract subjected to various 'wet chemical'
techniques before GC/MS analysis is possible.

The sample solution is injected into the GC inlet where it is vaporized and swept onto a
chromatographic column by the carrier gas (usually helium). The sample flows through the column
and the compounds comprising the mixture of interest are separated by virtue of their relative
interaction with the coating of the column (stationary phase) and the carrier gas (mobile phase). The
latter part of the column passes through a heated transfer line and ends at the entrance to ion source
(Fig. 1) where compounds eluting from the column are converted to ions.

41
Two potential methods exist for ion production. The most frequently used method is electron
ionisation (EI) and the occasionally used alternative is chemical ionisation (CI). For EI a beam of
electrons ionise the sample molecules resulting in the loss of one electron. A molecule with one
electron missing is called the molecular ion and is represented by M+. (a radical cation). When the
resulting peak from this ion is seen in a mass spectrum, it gives the molecular weight of the compound.
Due to the large amount of energy imparted to the molecular ion it usually fragments producing further
smaller ions with characteristic relative abundances that provide a 'fingerprint' for that molecular
structure. This information may be then used to identify compounds of interest and help elucidate the
structure of unknown components of mixtures. CI begins with the ionisation of methane (or another
suitable gas), creating a radical which in turn will ionise the sample molecule to produce [M+H]+
molecular ions. CI is a less energetic way of ionising a molecule hence less fragmentation occurs with
CI than with EI, hence CI yields less information about the detailed structure of the molecule, but does
yield the molecular ion; sometimes the molecular ion cannot be detected using EI, hence the two
methods complement one another. Once ionised a small positive is used to repel the ions out of the
ionisation chamber.

The next component is a mass analyser (filter), which separates the positively charged ions according
to various mass related properties depending upon the analyser used. Several types of analyser exist:
quadrupoles (Fig. 2), ion traps, magnetic sector, time-of-flight, radio frequency, cyclotron resonance
and focusing to name a few. The most common are quadrupoles and ion traps. After the ions are
separated they enter a detector the output from which is amplified to boost the signal. The detector
sends information to a computer that records all of the data produced, converts the electrical impulses
into visual displays and hard copy displays. In addtion, the computer also controls the operation of the
mass spectrometer.

2.LIQUID CHROMATOGRAPHY MASS SPECTROMETRY

42
Liquid chromatography-mass spectrometry (LC-MS, or alternatively HPLC-MS) is an analytical
chemistry technique that combines the physical separation capabilities of liquid chromatography (or
HPLC) 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. Generally its application is
oriented towards the specific detection and potential identification of chemicals in the presence of
other chemicals (in a complex mixture).

LIQUID CHROMATOGRAPHY

Scale

A major difference between traditional HPLC and the chromatography used in LC-MS is that in the
latter case the scale is usually much smaller, both with respect to the internal diameter of the column
and even more so with respect to flow rate since it scales as the square of the diameter. For a long
time, 1 mm columns were typical for LC-MS work (as opposed to 4.6 mm for HPLC). More recently
300 µm and even 75 µm capillary columns have become more prevalent. At the low end of these
column diameters the flow rates approach 100 nL/min and are generally used with nanospray sources

Flow splitting

When standard bore (4.6 mm) columns are used the flow is often split ~10:1. This can be beneficial by
allowing the use of other techniques in tandem such as MS and UV. However splitting the flow to UV
will decrease the sensitivity of spectrophotometric detectors. The Mass Spec on the other hand will
give improved sensitivity at flow rates of 200 μL/min or less. This is because the analyte ions must be
vaporised (nebulized) in order to become charged.

MASS SPECTROMETRY
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Mass analyzer

There are a lot of mass analyzers that can be used in LC/MS. Single Quadrupole, Triple Quadrupole,
Ion Trap, TOF (time of Flight) and Quadrupole-time of flight (Q-TOF).

Interface

Understandably the interface between a liquid phase technique which continuously flows liquid, and a
gas phase technique carried out in a vacuum was difficult for a long time. The advent of electrospray
ionization changed this. The interface is most often an electrospray ion source or variant such as a
nanospray source; however fast atom bombardment, thermospray and atmospheric pressure chemical
ionization interfaces are also used. Various deposition and drying techniques have also been used such
as using moving belts; however the most common of these is off-line MALDI deposition.

Applications

Pharmacokinetics

LC-MS is very commonly used in pharmacokinetic studies of pharmaceuticals. These studies give
information about how quickly a drug will be cleared from the hepatic blood flow, and organs of the
body. MS is used for this due to high sensitivity and exceptional specificity compared to UV (as long
as the analyte can be suitably ionised), and short analysis time.

The major advantage MS has is the use of tandem MS-MS. The detector may be programmed to select
certain ions to fragment. The process is essentially a selection technique, but is in fact more complex.
The measured quantity is the sum of molecule fragments chosen by the operator. As long as there are
no interferences or ion suppression, the LC separation can be quite quick. It is common now to have
analysis times of 1 minute or less by MS-MS detection, compared to over 10 mins with UV detection.

Proteomics

LC-MS is also used in the study of proteomics where again components of a complex mixture must be
detected and identified in some manner. The bottom-up proteomics LC-MS approach to proteomics
generally involves protease digestion and denaturation (usually trypsin as a protease, urea to denature
tertiary structure and iodoacetamide to cap cysteine residues) followed by LC-MS with peptide mass
fingerprinting or LC-MS/MS (tandem MS) to derive sequence of individual peptides. LC-MS/MS is
most commonly used for proteomic analysis of complex samples where peptide masses may overlap
even with a high-resolution mass spectrometer. Samples of complex biological fluids like human

44
serum may be run in a modern LC-MS/MS system and result in over 1000 proteins being identified,
provided that the sample was first separated on an SDS-PAGE gel or HPLC-SCX.

Drug development

LC-MS is frequently used in drug development at many different stages including Peptide Mapping,
Glycoprotein Mapping, Natural Products Dereplication, Bioaffinity Screening, In Vivo Drug
Screening, Metabolic Stability Screening, Metabolite Identification, Impurity Identification, Degradant
Identification, Quantitative Bioanalysis, and Quality Control.

3. ION MOBILITY SPECTROMETRY-MASS SPECTROMETRY

Ion mobility spectrometry-mass spectrometry (IMS-MS) is a method that combines the features of ion
mobility spectrometry and mass spectrometry to identify different substances within a test sample.

Ion mobility spectrometry/mass spectrometry (IMS/MS or IMMS) is a technique where ions are first
separated by drift time through some neutral gas under an applied electrical potential gradient before
being introduced into a mass spectrometer. Drift time is a measure of the radius relative to the charge
of the ion. The duty cycle of IMS (the time over which the experiment takes place) is longer than most
mass spectrometric techniques, such that the mass spectrometer can sample along the course of the
IMS separation. This produces data about the IMS separation and the mass-to-charge ratio of the ions
in a manner similar to LC/MS.

The duty cycle of IMS is short relative to liquid chromatography or gas chromatography separations
and can thus be coupled to such techniques, producing triple modalities such as LC/IMS/MS.

Data and analysis

On the one hand, ion mobility spectrometry was developed in the 70s and typically separates charged
particles on a millisecond scale. On the other hand, time-of-flight mass spectrometry was developed in
the 50s and typically separates charged particles on a microsecond scale. The combination of both
instruments was pioneered in the end of the 90s at Indiana University by David E. Clemmer and co-
workers

Instrumentation

The IMS-MS is composed of two major building blocks: the ion mobility spectrometer and the mass
spectrometer. Whereas the ion mobility spectrometer is usually made of a drift region at atmospheric
pressure or lower, the mass spectrometer is under a high vacuum.

45
Applications: The IMS-MS technique can be used in proteomics, for analyzing complex mixtures
of peptides.

MASS SPECTRA-REPRESENTATION

Mass spectrum of a peptide showing the isotopic distribution

APPLICATIONS OF MASS SPECTROMETRY

1.Isotope ratio MS: isotope dating and tracking

46
Mass spectrometer to determine the 16O/18O and 12C/13C isotope ratio on biogenous carbonate

Mass spectrometry is also used to determine the isotopic composition of elements within a sample.
Differences in mass among isotopes of an element are very small, and the less abundant isotopes of an
element are typically very rare, so a very sensitive instrument is required. These instruments,
sometimes referred to as isotope ratio mass spectrometers (IR-MS), usually use a single magnet to
bend a beam of ionized particles towards a series of Faraday cups which convert particle impacts to
electric current. A fast on-line analysis of deuterium content of water can be done using Flowing
afterglow mass spectrometry, FA-MS. Probably the most sensitive and accurate mass spectrometer for
this purpose is the accelerator mass spectrometer (AMS). Isotope ratios are important markers of a
variety of processes. Some isotope ratios are used to determine the age of materials for example as in
carbon dating. Labelling with stable isotopes is also used for protein quantification. (see Protein
quantitation below)

2.Trace gas analysis

Several techniques use ions created in a dedicated ion source injected into a flow tube or a drift tube:
selected ion flow tube (SIFT-MS), and proton transfer reaction (PTR-MS), are variants of chemical
ionization dedicated for trace gas analysis of air, breath or liquid headspace using well defined reaction
time allowing calculations of analyte concentrations from the known reaction kinetics without the need
for internal standard or calibration.

3.Atom probe

An atom probe is an instrument that combines time-of-flight mass spectrometry and field ion
microscopy (FIM) to map the location of individual atoms.

47
4.Pharmacokinetics

Pharmacokinetics is often studied using mass spectrometry because of the complex nature of the
matrix (often blood or urine) and the need for high sensitivity to observe low dose and long time point
data. The most common instrumentation used in this application is LC-MS with a triple quadrupole
mass spectrometer. Tandem mass spectrometry is usually employed for added specificity. Standard
curves and internal standards are used for quantitation of usually a single pharmaceutical in the
samples. The samples represent different time points as a pharmaceutical is administered and then
metabolized or cleared from the body. Blank or t=0 samples taken before administration are important
in determining background and insuring data integrity with such complex sample matrices. Much
attention is paid to the linearity of the standard curve; however it is not uncommon to use curve fitting
with more complex functions such as quadratics since the response of most mass spectrometers is less
than linear across large concentration ranges.

There is currently considerable interest in the use of very high sensitivity mass spectrometry for
microdosing studies, which are seen as a promising alternative to animal experimentation.

5. Protein characterization

Mass spectrometry is an important emerging method for the characterization of proteins. The two
primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted
laser desorption/ionization (MALDI). In keeping with the performance and mass range of available
mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are
ionized by either of the two techniques described above, and then introduced to a mass analyser. This
approach is referred to as "top-down" strategy of protein analysis. In the second, proteins are
enzymatically digested into smaller peptides using proteases such as trypsin or pepsin, either in
solution or in gel after electrophoretic separation. Other proteolytic agents are also used. The collection
of peptide products are then introduced to the mass analyser. When the characteristic pattern of
peptides is used for the identification of the protein the method is called peptide mass fingerprinting
(PMF), if the identification is performed using the sequence data determined in tandem MS analysis it
is called de novo sequencing. These procedures of protein analysis are also referred to as the "bottom-
up" approach.

6.Space exploration

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As a standard method for analysis, mass spectrometers have reached other planets and moons. Two
were taken to Mars by the Viking program. In early 2005 the Cassini-Huygens mission delivered a
specialized GC-MS instrument aboard the Huygens probe through the atmosphere of Titan, the largest
moon of the planet Saturn. This instrument analyzed atmospheric samples along its descent trajectory
and was able to vaporize and analyze samples of Titan's frozen, hydrocarbon covered surface once the
probe had landed. These measurements compare the abundance of isotope(s) of each particle
comparatively to earth's natural abundance.. Also onboard the Cassini-Huygens spacecraft is an ion
and neutral mass spectrometer which has been taking measurements of Titan's atmospheric
composition as well as the composition of Enceladus' plumes.

Mass spectrometers are also widely used in space missions to measure the composition of plasmas. For
example, the Cassini spacecraft carries the Cassini Plasma Spectrometer (CAPS), which measures the
mass of ions in Saturn's magnetosphere.

7.Respired gas monitor

Mass spectrometers were used in hospitals for respiratory gas analysis beginning around 1975 through
the end of the century. Some are probably still in use but none are currently being manufactured.

Found mostly in the operating room, they were a part of a complex system in which respired gas
samples from patients undergoing anesthesia were drawn into the instrument through a valve
mechanism designed to sequentially connect up to 32 rooms to the mass spectrometer. A computer
directed all operations of the system. The data collected from the mass spectrometer was delivered to
the individual rooms for the anesthesiologist to use.

This magnetic sector mass spectrometer's uniqueness may have been the fact that a plane of detectors,
each purposely positioned to collect all of the ion species expected to be in the samples, allowed the
instrument to simultaneously report all of the patient respired gases. Although the mass range was
limited to slightly over 120 u, fragmentation of some of the heavier molecules negated the need for a
higher detection limit.

References

1. Sparkman, O. David (2000). Mass spectrometry desk reference. Pittsburgh: Global View Pub.
ISBN 0-9660813-2-3.
49
2. "Definition of spectrograph." Merriam Webster. Accessed 13 June 2008.
3. KM Downard (2007). "William Aston - the man behind the mass spectrograph". European
Journal of Mass Spectrometry 13 (3): 177–190. doi:10.1255/ejms.878.
4. Harper, Douglas. "Spectrum." Online Etymology Dictionary. Nov. 2001. Accessed 07-12-
2007. Note: This part of the article only makes descriptive claims about the information found
in the primary source, the accuracy and applicability of which is easily verifiable by any
reasonable, educated person without specialist knowledge. (See WP:PSTS)
5. Squires, Gordon (1998). "Francis Aston and the mass spectrograph". Dalton Transactions:
3893–3900.
6. KM Downard (2007). "Francis William Aston - the man behind the mass spectrograph".
European Journal of Mass Spectrometry 13 (3): 177–190
7. Thomson, J.J. (1913). Rays Of Positive Electricity and Their Application to Chemical Analysis.
London: Longman's Green and Company.
http://www.archive.org/details/RaysOfPositiveElectricity.
8. Siri, William (August 1947). "Mass spectroscope for analysis in the low-mass range". Review
of Scientific Instruments 18 (8): 540–545.
9. Price, Phil (1991). "Standard definitions of terms relating to mass spectrometry. A report from
the Committee on Measurements and Standards of the American Society for Mass
Spectrometry". Journal of the American Society for Mass Spectrometry 2 (4): 336–348.
10. J.J., Thomson (1913). Rays Of Positive Electricity and Their Application to Chemical Analysis.
London: Longman's Green and Company.
http://www.archive.org/details/RaysOfPositiveElectricity.
11. Measuring Mass: From Positive Rays to Proteins by Michael A. Grayson (Editor) (ISBN 0-
941901-31-9)
12. Carl-Ove Anderson, Acta. Chem. Scand. 1958, 12, 1353
13. Mass Spec Anniversary, Chemical & Engineering News, 87, 6 (9 Feb. 2009), p. 4
14. A. P. Bruins (1991). "Mass spectrometry with ion sources operating at atmospheric pressure".
Mass Spectrometry Reviews 10 (1): 53–77.
15. John S Cottrell, Roger J Greathead (1986). "Extending the Mass Range of a Sector Mass
Spectrometer". Mass Spectrometry Reviews 5: 215–247.
16. Wollnik, H. (1993). "Time-of-flight mass analyzers". Mass Spectrometry Reviews 12: 89.
17. Paul W., Steinwedel H. (1953). "Ein neues Massenspektrometer ohne Magnetfeld". Zeitschrift
für Naturforschung A 8 (7): 448–450.
18. R. E. March (2000). "Quadrupole ion trap mass spectrometry: a view at the turn of the
century". International Journal of Mass Spectrometry 200 (1-3): 285–312

50
19. Schwartz, Jae C.; Michael W. Senko and John E. P. Syka (June 2002). "A two-dimensional
quadrupole ion trap mass spectrometer". Journal of the American Society for Mass
Spectrometry (Elsevier Science B.V.) 13 (6): 659–669.
20. M. B. Comisarow and A. G. Marshall (1974). "Fourier transform ion cyclotron resonance
spectroscopy". Chemical Physics Letters 25 (2): 282–283. doi:10.1016/0009-2614(74)89137-2.
21. Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. (1998). "Fourier transform ion cyclotron
resonance mass spectrometry: a primer". Mass Spectrometry Reviews 17 (1): 1–34.
22. Q. Hu, R. J. Noll, H. Li, A. Makarov, M. Hardman and R. G. Cooks (2005). "The Orbitrap: a
new mass spectrometer". Journal of Mass Spectrometry 40 (4): 430–443
23. F. Dubois, R. Knochenmuss, R. Zenobi, A. Brunelle, C. Deprun and Y. L. Beyec (1999). "A
comparison between ion-to-photon and microchannel plate detectors". Rapid Communications
in Mass Spectrometry 13 (9): 786–791
24. M. A. Park, J. H. Callahan and A. Vertes (1994). "An inductive detector for time-of-flight mass
spectrometry". Rapid Communications in Mass Spectrometry 8 (4): 317–322.
25. Robert K. Boyd (1994). "Linked-scan techniques for MS/MS using tandem-in-space
instruments". Mass Spectrometry Reviews 13 (5-6): 359–410.
26. Eiceman, G.A. (2000). Gas Chromatography. In R.A. Meyers (Ed.), Encyclopedia of
Analytical Chemistry: Applications, Theory, and Instrumentation, pp. 10627. Chichester:
Wiley. ISBN 0-471-97670-9
27. Verbeck, GF and Ruotolo, BT and Sawyer, HA and Gillig, KJ and Russell, DH (2002). "A
fundamental introduction to ion mobility mass spectrometry applied to the analysis of
biomolecules". J Biomol Tech 13 (2): 56–61. PMC: 2279851.
http://jbt.highwire.org/cgi/content/abstract/13/2/56.
28. L. M. Matz, G. R. Asbury and H. H. Hill (2002). "Two-dimensional separations with
electrospray ionization ambient pressure high-resolution ion mobility spectrometry/quadrupole
mass spectrometry". Rapid Communications in Mass Spectrometry 16 (7): 670–675
29. Rena A. Sowell, Stormy L. Koeniger, Stephen J. Valentine, Myeong Hee Moon and David E.
Clemmer (2004). "Nanoflow LC/IMS-MS and LC/IMS-CID/MS of Protein Mixtures". Journal
of the American Society for Mass Spectrometry 15 (9): 1341–1353
30. Tureček, František; McLafferty, Fred W. (1993). Interpretation of mass spectra. University
Science Books, Sausalito, ISBN 0-935702-25-3
31. Mistrik, R.(2004). A New Concept for the Interpretation of Mass Spectra Based on a
Combination of a Fragmentation Mechanism Database and a Computer Expert System.in
Ashcroft, A.E., Brenton, G., Monaghan,J.J. (Eds.), Advances in Mass Spectrometry, Elsevier,
Amsterdam, vol. 16, pp. 821.

51
32. Hsieh, Yunsheng (2006). "Systems for Drug Metabolism and Pharmacokinetic Screening, Y.
Hsieh and W.A. Korfmacher, Current Drug Metabolism". Current Drug Metabolism 7 (5):
479–489.
33. Covey, T.R.; Lee, E.D.; Henion, J.D. (1986). "Mass Spectrometry for the Determination of
Drugs in Biological Samples". Anal. Chem. 58: 2453–2460..
34. Covey, Tom R. (Feb 1985). "Mass Spectrometry Determination of Drugs and Their
Metabolites in Biological Fluids". Anal. Chem. 57 (2): 474–81.
35. S. Petrie and D. K. Bohme (2007). "Ions in space". Mass Spectrometry Reviews 26 (2): 258–
280.
36. "Cassini Plasma Spectrometer". Southwest Research Institute. http://caps.space.swri.edu/.
Retrieved 2008-01-04.
37. Riker JB, Haberman B (1976). "Expired gas monitoring by mass spectrometry in a respiratory
intensive care unit". Crit. Care Med. 4 (5): 223–9.
38. J. W. W. Gothard, C. M. Busst, M. A. Branthwaite, N. J. H. Davies and D. M. Denison (1980).
"Applications of respiratory mass spectrometry to intensive care". Anaesthesia 35 (9): 890–
895..

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