mass spectroscopy presentation.pptx

The Basics and Applications of Mass
Spectroscopy
Advanced Instrumentation Techniques
Presented By:
Kushkiwala Asefa M.arif
Guided by:
Dr. Amit. K. Joshi (M.pharm., Ph.D.)
( Associate professor &
Head of Quality Assurance )

Table of Content
1. Introduction
2. Principle
3. Instrumentation
4. Applications of Mass Spectrometry
5. Conclusion
6. Reference

1.Introduction
1. Mass-Spetrometry is a modern analytical technique which is very applicable
in identification of unknown compound in the sample, and also for structure
elucidation.
2. It is a microanalytical technique requiring only a few nanomoles of the sample
to obtain characteristic information pertaining to the structure and molecular
weight of analyte.
3. In mass spectrometry, one generates ions from a sample to be analyzed.
These ions are then separated and quantitatively detected. Separation
is achieved on the basis of different trajectories of moving ions with
different mass/charge (m/z) ratios in electrical and/or magnetic
fields.
4. It involves the production and separation of ionised molecules and their ionic
decompositon product and finally the measurement of the relative abundance of
different ions produced. It is, thus a destructive technique in that the sample is
consumed during analysis.
5. Mass-spectrometry has evolved from the experiments and studies early in the
20th century that tried to explain the behavior of charged particles in magnetic
and electrostatic force fields. Well-known names from these early days are J. J.
Thomson used mass spectrum to demonstrate the existence of neon-22 in a
sample of neon-20

2. Principle of Mass Spectroscopy:
 In the simplest mass spectrometer , organic molecules bombarded with
electrons and converted to highly energetic charged ions (molecular ions or
parent ions), which can break up into smaller ions (fragment ions, or
daughter ions); the loss of an electron a molecule leads to a radical cation,
and we can represent this process as;
M → M.+
 The molecular ion M.
+ commonly decomposes to a pair of fragments,
which may be either a radical plus an ion, or a small molecule plus a radical
cation. Thus,
M → m1
+ + m2
. or m1
. + m2
+
 The molecular ions, the fragment ions and the fragment radical ions are
separated by deflection in a variable magnetic field according to their mass
and charge ,and generate a current (the ion current) at the collector in
proportion to their relative abundances. A mass spectrum is a plot relative
abundance against the ratio mass/charge (the m/z value), For singly charged
ions, the lower the mass the more easily is the ion in the magnetic field.

 This Figure shows a simplified line-
diagram representation of the mass
spectrum of 2-methylpentane
(C6H14). The most abundant ion has an
m/z value of 43 (corresponding to
C3H7+), showing that the most favored
point of rupture occurs between Cx, and
Cy : this most abundant ion (the base
peak) is given an arbitrary abundance of
100, and all other intensities are
expressed as a percentage of this (relative
abundances). The small peak at m/z 86
is obviously the molecular ion. The peaks
at m/z 15, 29 and 71 correspond to CH3+.
C2H5+ and C5H11+, respectively, etc.: the
fragment ions arise from the rupture of
the molecular ion, either directly or
indirectly.
C6H14
(Bombardment)
C6H14
+
(Molecular ion)

General modes of
fragmentation:
Fragmentation of the molecular ion takes
place in following modes:
Simple cleavage
1. Homolytic cleavage
2. Heterolytic cleavage
3. Retro Diels-Alder reaction
 Rearrangement reactions
accompanied by transfer of
atoms.
1.Scrambling
2.Mc Lafferty rearrangement
3. Elimination
Fragmentation process:
 Bombardment of molecules by an electron
beam with energy between 10-15ev usually
results in the ionization of molecules by
removal of one electron (Molecular
ion formation).
 When the energy of electron beam is
increased between 50-70ev, these molecular
ions acquire a high excitation resulting in
their break down into various fragments.
This process is called “ Fragmentation
process”.
Types of Ions:
1.Molecular ion or Parent ion.
2.Fragment ions..
3.Rearrangement ions. result of intramolecular atomic rearrangement during fragmentation.
4.Multicharged ions ions may also exist with two or three charges instead of usual single charge in
the mass spectrum. These are known as doubly or triply charged ions. They are created as follows:
M+° + e- → M++ + 3e
5.Negative ions 1.AB + e → A+ + B— 2.AB + e → AB—
6.Metastable ions Fragment of a parent ion will give rise to a new ion (daughter) plus
either a neutral molecule or a
M1+ M2+ + non charged particle

3. Instrumentation:
3.1 Classification of Mass Spectrometer:
 Mass spectrometers for structure elucidation can be classified according to
the method of separating the charged particles:
A. Magnetic Field Deflection
1. Magnetic Field Only (Unit Resolution)
 The gas stream from the
inlet system enters the
ionization chamber in which
it is bombarded at right
angles by an electron beam
emitted from a hot filament.
Positive ions produced
by interaction with the
electron beam are
forced through the first
accelerating slit by a
weak electrostatic field.
A strong electrostatic field
then accelerates the ions to
their final velocities. To
obtain a spectrum, the
applied magnetic field is
increased, bringing
successively heavier ions
into the collector slit.

2. Double Focusing (Electrostatic Field and Magnetic Field, High
Resolution)
 The introduction of an
electrostatic field after (or
before) the magnetic field
permits high resolution so that
the mass of a particle can be
obtained to four decimal places.
This figure shows a double-
focusing instrument. Ions
generated in the source are
accelerated toward the analyzer.
The magnetic field provides
directional focusing. The path of
the positive ion is again curved
by the electric field applied
perpendicular to the flight path
of the ions. This double focusing
provides resolution as high as
60,000.

B. Quadrupole Mass Spectrometry
1.Quadrupole Mass Filter (B.1)
 This mass filter uses four voltage-carrying rods (the "quadrupole“). Ions entering from one end
travel with constant velocity in the direction parallel to the poles (z direction), but acquire
complex oscillations in the x and y directions by application of both a direct current (dc)
voltage (Vdc) and a radiofrequency (Vrf) voltage (V) to the poles. There is a “stable
oscillation" that allows a particular ion to pass from one end of the quadrupole to the other
without striking the poles; this oscillation is dependent on the m/z ratio of an ion. Therefore,
ions of only a single m/z value will traverse the entire length of the filter at a given set of
conditions. All other ions will have unstable oscillations and will strike the poles and be lost.
Mass scanning is carried out by varying each of the Rf and dc frequencies while keeping their
ratios constant.

2. Quadrupole Ion Storage (Ion Trap) (B.2)
 Essentially, the ion storage trap is a spherical configuration of the linear
quadrupole mass filter. The operations, however, differ in that the linear filter
passes the sorted ions directly through to the detector, whereas the ion trap retains
the unsorted ions temporarily within the trap. They are then released to the
detector sequentially by scanning the electric field. These instruments are compact
(benchtop), relatively inexpensive, convenient to use, and very sensitive.

C. Time of Flight MassSpectrometer
 In the time-of-flight (TOF) mass spectrometers, all singly charged particles subjected to a
potential difference V attain the same translational energy in electron volts (eV). Thus lighter
particles have the shorter TOF over a given distance. The accelerated particles are passed into a
field-free region where they are separated in time by their m/z values and collected. Since
arrival times between successive ions can be less than 10-7s, fast electronics are necessary for
adequate resolution. Time-of flight devices are used with sophisticated ionizing methods ( FAB,
Laser desorption, and plasma desorption ).
 Positive ions are produced periodically
by bombardment of the sample with
brief pulses of electrons, secondary
ions, or laser-generated photons. The
ions produced in this way are then
accelerated into a field-free drift
tube by an electric field pulse of
103 to 104V . Separation of ions by
mass occurs during the transit of the
ions to the detector located at the end
of the tube. Because all ions entering
the tube have the same kinetic energy,
their velocities in the tube vary
inversely with their masses , with the
lighter particles arriving at the
detector earlier than the heavier ones.
The flight time tf is given by
tf=
Where L=Distance from source

D. FT-ICR (Fourier Transform-Ion Cyclotron Resonance) (D) (Also termed
FT-MS)
 As was true with infrared and nuclear magnetic resonance instruments. Fourier transform mass
spectrometers provide improved signal-to-noise ratios, greater speed, and higher
sensitivity and resolution .Commercial Fourier transform mass spectrometers appeared on
the market in the early 1980s and are now offered by several manufacturers. The heart of a
Fourier transform instrument is an ion trap within which ions can circulate in well
defined orbits for extended periods. Such cavities are constructed to take advantage of a
phenomenon known as ion cyclotron resonance.
 When a gaseous ion drifts into or is formed in a strong magnetic field, its motion becomes
circular in a plane perpendicular to the direction of the field. The angular frequency of this
motion is called the cyclotron frequency, ɷc Equation can be rearranged and solved for v/r,
which is the cyclotron frequency in radians per second.
ɷc = v/r = zeB/m

E. Tandem Mass Spectrometer
 Tandem mass spectrometry, sometimes called mass spectrometry-mass spectrometry
(MS/MS), is a method that allows the mass spectrum of preselected and fragmented ions to be
obtained .Here, an ionization source, often a soft ionization source, produces ions and some
fragments. These are then the input to the first mass analyzer, which selects a particular ion
called the precursor ion and sends it to the interaction cell. In the interaction cell, the
precursor ion can decompose spontaneously react with a collision gas, or interact with an
intense laser beam to produce fragments, called product ions. These ions are then mass
analyzed by the second mass analyzer and detected by the ion detector.

 Tandem mass spectrometry has been implemented in a number of ways. These
can be classified as tandem in space and tandem in time.
 A common use of tandem-MS is the analysis of biomolecules, such as protiens and
peptides.
 Types:
(1) Tandem in time
 In the same space, fragmentation of selected ions is carried out subsequently
number of time.

(2)Tandem-in-Space
 Tandem MS in space uses the coupling of two instrument components
which measure the same mass spectrum range but with a controlled
fractionation between them in space, while tandem MS in time involves
the use of an ion trap.

 Product ion scanning.
The precursor ion is focussed in Quadrupole 1 (MS1) and transferred into
Quadrupole 2 - the collision cell. Here it undergoes CID and fragments. The
fragments are then transferred into Quadruole 3 (MS2) which is scanned to produce
a spectrum of fragments - the product ion spectrum. This results in a specific MS/MS
spectrum of one single precursor ion independently of any other ions present in the
spectrum and is the most commonly employed method.
 Precursor ion scanning.
Quadrupole 3 is held static at the m/z of a specific product ion only and quadrupole 1
is scanned across the desired m/z range. This experiment results in a spectrum of
precursor ions that produce that particular product ion during CID fragmentation.
This experiment is used in metabolite profiling to search for common structural
cores (the product ion) - that maybe characteristic of a particular compound class (in
a complex mixture).
 Neutral loss scanning.
Quadrupole 1 is scanned as well as Quadrupole 3. The combination of scanning is
used to detect precursor ions that fragment via a specific neutral loss during CID
fragmentation. This mode of operation is also often used in metabolite profiling to
search for common functional groups (the neutral lost) - that maybe characteristic of
a particular compound class (in a complex mixture).

 Various Dissociative Interactions in the Interaction Cell:
(1) Collisionally activated dissociation (CAD)
 fragmentation can be induced by adding a collision gas(He gas) to the
interaction cell so that interaction with precursor ions occurs, leading to
decomposition into product ions. In this case, the cell is called a collision cell,
and the interactions are termed collisionally activated dissociation (CAD)
or alternatively collision-induced dissociation (CID).
(2) Electron-capture dissociation (ECD)
 precursor ions capture a low-energy electron to produce an intermediate that
rapidly dissociates. In some cases, a background gas is added to aid in the
dissociation process.
(3) Photo-induced dissociation (PID)
 to stimulate decomposition of precursor ions. In nearly all cases, an intense
laser beam is used in the interaction cell to promote the dissociation.
(4) surface-induced dissociation (SID)
 in which precursor ions interact with a surface to induce dissociation. Ions have
been reflected off cell walls or trapping plates to increase their internal energy
and promote dissociation.
 A difficulty with PID is that the ion beam and photon beam must overlap in the
interaction region for a time long enough for absorption and bond rupture to
occur.

3.2 Components of Mass Spectrometer
 Mass spectrometer consists of following basic components and a schematic view of the major
components of a mass spectrometer is presented below.
1. Sample inlet
The purpose of the inlet system is to permit introduction of a representative sample into the
ion source with minimal loss of vacuum. Most modern mass spectrometers are equipped
with several types of inlets to accommodate various kinds of samples; these include
1. Batch inlets,
2. Direct probe inlets,
3. Chromatographic inlets and
4. Capillary electrophoretic inlets.
2. Ionization Source / Ionization Techniques
 The first and important step to obtained mass spectrum is to ionize the sample under
investigation.
 The minimum energy require to ionize an atom or a molecule is called ionization potential.
 The common technique used for formation of ions is by bombardment of high energy beam of
electron.
 The bombardment of electron is produced from an electrically heated tungsten filament.
 Molecular ions are formed only when the energy of electron beam reaches to 10- 15 eV.

 Because the energy require for removing one electron from the neutral parent
molecule is usually 10 eV.
 And fragmentation of ions is only happen when energy of beam reaches to 70 eV.
Because if energy is high around 70eV, then additional energy is consumed in
fragmenting the parent or molecular ion. This result into fragmented ions or
daughter ion

1. Electron Impact Ionization
 Organic molecules react on electron impact in two ways: either an electron is captured by
molecule, giving a radical anion, or an electron is removed from the molecule, giving a
radical cation:
M + e- → M+ + 2e-
2. Chemical Ionization:
 In chemical ionization, gaseous atoms of the sample (from either a batch inlet or a heated
probe) are ionized by collision with ions produced by electron bombardment of an
excess of a reagent gas. Usually, positive ions are used, but negative ion chemical
ionization is occasionally used with analytes that contain very electronegative atoms.
Chemical ionization is probably the second-most common procedure for producing ions for
mass spectrometry.

 Soft ionization method (more controlled) / some fragmentation observed
 Indirect ionization of sample
 Collisions between sample & gas ions cause proton transfers / produces [M+H]+
ions, not M+ ions (so parent is M+1)
 More controlled than EI / reduced fragmentation / greater sensititivty / typically
~5 eV energy transfer
 Good for molecular weight determination
 Provides less information about structure
M + H3+ [M+H]+ + H2
M + CH5+ [M+H]+ + CH4
M + C2H5+ [M+C2H5]+
M + (CH3)3C+ [M+H]+ + (CH3)2=CH2
M + (CH3)3C+ [M+C(CH3)3]+
M + NH4+ [M+H]+ + NH3+
 Ionization methods include proton transfer and adduct formation
 Results in formation of "quasimolecular" ion

3. Field Desorption Methods
 In field desorption, a multi-tipped
emitter similar to that used in field
ionization sources is employed. In this
case, the electrode is mounted on a
probe that can he removed from the
sample compartment and coated with
a solution of the sample. After the
probe is reinserted into the sample
compartment, ionization again takes
place by applying a high voltage to this
electrode.
4. MALDI:
 The abbreviation for "Matrix Assisted
Laser Desorption/Ionization The simple
for MALDI is uniformly moved in quantity of
matrix The matrix absorbs the
ultraviolet light (nitrogen laser light,
wavelength 337 m) a converts it to heat
energy. A small part of the matrix (down
to 100 nm from the top surface of the Analyte
in the diagram) heats rapidly (in several
nano seconds) and vaporized, together
with the sample.

 Soft ionization method
 Sample is mixed with a condensed phase matrix that contains a chromophore
 Mixture is ionized with a laser à proton tranfer from matrix to sample
 Charged molecules are ejected from matrix
 Little excess energy → little fragmentation
 Good for large molecules (proteins, polymers, carbohydrates)
[ Table : Example of various MALDI matrix ]

5. FAB
 Fast atom bombardment spectrometry can be considered to be an extension of secondary
ion mass spectrometry. Fast atom bombardment (FAB) sources, also called liquid secondary-
ion sources, have assumed a major role in the production of ions for mass spectrometric
studies of polar high-molecular-mass species.
6. Electrospray Ionization :
 Electrospray ionization-mass spectrometry (ESI/MS). which was first described in 1984 has now
become one of the most important techniques for analyzing biomoleeules, such as
polypeptides, proteins, and oligonucleotides, having molecular weights of 10,000
Da or more.
 Soft ionization method
 Sample mixed with a condensed phase matrix (glycerol) protects sample from excess
energy
 Mixture ionized by bombarding with beam of high energy atoms à Xe or Ar (6-10
keV)
 Ionization from protonation ([M+H]+) or cation attachment ([M+Na]+)
 High resolution, exact mass determination is possible
 Soft Ionization method
 Does not require vacuum pressures
 Sample solution pumped through a narrow, stainless steel capilliary à aerosol of
charged droplets

3 . Mass analyzer
In the mass analyzer ions are separated based on their mass to charge
(m/z) ratios applying electric field (electrostatic analyzer) or
magnetic field (magnetic analyzer) or Bo The ions are analyzed by
measuring the electric field or both required for getting peak in the spectrum,
or by measuring the time they take to travel a fixed distance.
 Solvent evaporates until
electrostatic repulsion
within droplets too great /
Coulombic explosion
 Sample ions released into
vapor phase [M+H]+,
[M+Na]+
 Good for large molecules
(proteins, polymers,
carbohydrates)

Type of analyzer Symbol Principle of separation
Electric sector E or ESA (Kinetic energy)
Magnetic sector B (Momentum)
Quadrupole Q m/z (trajectory stability)
Ion trap IT m/z (resonance frequency)
Time-of-flight TOF Velocity (flight time)
Fourier transform ion cyclotron resonance FTICR m/z (resonance frequency)
Fourier transform or bitrap FT-OT m/z (resonance frequency)
Types of analyzers used in mass spectrometry.
 All mass analyzers use static or dynamic electric and magnetic fields that can be
alone or combined.
 Once the gas-phase ions have been produced, they need to be separated according to
their masses, which must be determined.
 The physical property of ions that is measured by a mass analyzer is their mass-to-
charge ratio (m/z) rather than their mass alone.

4. Detectors:
 A detector converts the beam of ions into a usable signal through the generation of
electric current which is proportional to the ion abundance.
1. Faraday cup collector:
 The basic principle involves that the incident ion strike the dynode surface which
emits electron and induces a current which is amplified and recorded.
 A Faraday cup is made of a metal cup or cylinder with a small orifice.
 Ions reach the inside of the cylinder and are neutralized by either accepting or
donating electrons as they strike the walls. This leads to a current through the
resistor. The discharge current is then amplified and detected. It provides a
measure of ion abundance.
 The dynode electrode ids made of secondary emitting material like CsSb, GaP,
BeO.
2. Electron multiplier:
 Electron multiplier are the most commonely used detector in MS. Specially when
positive and negative ions needs to be detected on the same instrument.
 Dynodes made up of copper-beryllium which transduces the initial ion current,
and electron emitted by first dynode are focused magnetically from dynode to the
nest
 Final current is amplifies more than millions times
3. Photographic plate detection:

5. Read out system :
6. Vacuum system :
 For the operation of mass spectrometer, the ion source, the mass analyzer, and the
detector must be kept under high vacuum conditions of 10-6 to 10-7 torr. Both
the speed at which instrument is operational after cleaning or opening to
atmospheric pressure and the efficiency of maintaining a high vacuum are related to
the capacity and speed of vacuum system.
 Most systems use a combination of oil diffusion pumps to maintain high vacuum and
hacking rotary pups to reduce the initial pressure to approx. 0.001 torr. However
oil diffusion pumps are being replaced more and more turbo molecular pumps.
 Electrical detection of mass spectrum allows operation of the ion-collection system
in one of the several modes. Scanning the mass spectrum across the detector is an
extremely inefficient way to collect information and is seldom done.
 Resolution generally suffers in peak switching because the slits must be widened to
allow for slight inaccuracies in field settings while still viewing a portion of the mass
spectrum that includes the mass number of interest.
 In the selective-ion mode the instrument simply monitors a single mass number to
achieve low limits of detection by long-term integration of the signal. On the modern
mass spectrometers the data are digitalized and collected on magnetic tape or stored
in the memory of a computer for subsequent processing. Such a system permits
rapid accumulation of the wealth of data generated. On request, the dedicated
micrometer reconstructs the mass spectrum.

5.Applications of Mass Spectrometry
 Mass spectrometry (MS) can ionize a sample and measure the mass-to-charge (m/z)
ratios of the resulting ions. With the combination of the mass spectrometer,
computer technology, and the software, the range of applications of MS becomes
more and more extensive.
(1). Mass Spectrometry in Proteomics
 To precisely determine the molecular mass of peptides and proteins and the
sequences.
 MS-based protein identification methods, including de novo sequencing and
peptide mass fingerprinting (PMF) database searching
 There are some methods commonly used for protein quantification, such as
ITRAQ (isobaric tags for relative and absolute quantitation), SILAC (stable
isotope labeling with amino acids in cell culture), ICAT (isotope-coded affinity
tags), label-free quantification and so on.
 Post-translational modifications (PTMs) are chemical alterations to protein
structure, which are typically catalyzed by substrate-specific enzymes. There are
various types of PTM, including phosphorylation, acetylation,
glycosylation, and so on.
 MS is considered a key technology for the protein modifications analysis because it
can provide universal information about protein modifications without a priori
knowledge and location of the modifying sites

(2). Mass Spectrometry in Metabolomics
 MS-based metabolomics studies the effect of drugs, toxins, and various diseases on
metabolite levels, to trace metabolic pathways and measure fluxes.
(3). Mass Spectrometry Imaging
 Mass spectrometry imaging (MSI) is a technology to visualize the spatial distribution
of molecules.
(4). Phytochemical analysis
 Mass spectroscopy is widely employed in phytochemical analysis due to its capability to
identify and measure metabolites having very low molecular weight at very low
concentration ranges below nanogram per milliliter (ng/mL). Therefore, it is considered as
trace analysis methodology.
 Mass spectrometers like quadrupole or quadrupole-time-of-flight (Q-TOF) are
frequently employed in combination along with gas chromatographic system. Several
phytoconstituents are volatile and thermolabile, and they can be analyzed by electrospray
ionization (ESI) and matrix-assisted laser desorption ionization (MALDI).
 Fourier transform ion cyclotron resonance (FT-ICR), orbitrap and TOF are emerged as high-
performance mass analyzers that are able to screen metabolites with fraction of seconds due to
their high resolution.
 Combination of TOF with one (Q-TOF) or two quadrupoles (Qq-TOF) is emerged as hybrid
mass spectrometers that are able to cover unlimited mass range with high scan rates up to 106
u/s and high resolving power.

 Analytes having
high molecular
weight and
temperature
sensitive can be
efficiently
analyzed by HPLC
coupled with
atmospheric
pressure
ionization-mass
spectrometer
(API-MS).
 Some of the recent
research articles
depicting the
application of
mass spectrometry
for the
phytochemical
analysis are listed
in Table .

(5) Structural Information from Fragmentation Patterns
 Systematic studies of fragmentation patterns for pure substances have led to rational
guidelines to predict fragmentation mechanisms and a series of general rules helpful
in interpreting spectra
(6) Analysis of Mixtures by Hyphenated Mass Spectral Method
 mass spectrometers are coupled with various efficient separation devices in so-called
hyphenated methods.
1. Chromatography-Mass Spectrometry
 Gas chromatography-mass spectrometry (GC/MS) has become one of the most
powerful tools available for the analysis of complex organic and biochemical
mixtures.
2. Capillary Electrophoresis-Mass Spectrometry
 In most of the applications reported to date, the capillary effluent is passed directly
into an electrospray ionization device. and the products then enter a quadrupole
mass filter for analysis.
3. Applications of Tandem Mass Spectrometry
 For some complex mixtures the combination of GC or LC and MS does not provide
enough resolution. In recent years, it has become feasible to couple chromatographic
methods with tandem mass spectrometers to form GC/MS/MS and LC /MS/MS
systems.

 To date, tandem mass spectrometry has been applied to the qualitative and
quantitative determination of the components of a wide variety of complex materials
encountered in nature and industry.
 Some examples include the identification and determination of drug metabolites,
insect pheromones, alkaloids in plants, trace contaminants in air.
 polymer sequences petrochemicals, polychlorinated biphenyls, prostaglandins, diesel
exhausts and odors in air.
(7) Retro Diels-Alder fragmentation
 EI-MS give idea about the Retro Diels-Alder fragmentation by providing the
mass spectrum of their fragment ion and molecular ion, so we can get idea about the
product ion along with their mass spectrum.
 Retro Diels-Alder Reaction Spectra of 4-4 methylcyclohexene:

(8) Detection of Environmental contaminants
 Environmental toxins in air, water and soil are routinely analyzed via mass spectrometry
because this technology offers unprecedented resolution and detection of trace contaminants.
(9) Pharma & Biopharma
 Pharmaceuticals and biopharmaceuticals must undergo rigorous processes of testing,
production and verification. MS systems and associated workflows expedite these processes by
aiding in the discovery and characterization of candidate compounds, as well as their safety and
efficacy testing.
(10) Structure Elucidation
1. The Rule of Thirteen ( M13 ) :
 A useful method for generating possible molecular formulas for a given molecular
mass is the Rule of Thirteen.
 As a first step in the Rule of Thirteen, we generate a base formula, which contains
only carbon and hydrogen. The base formula is found by dividing the molecular
mass, M, by 13 (the mass of one carbon plus one hydrogen). This calculation provides
a numerator, n, and a remainder, r.
M/13 = n + r/13
The base formula thus becomes
CnHn+r
 which is a combination of carbons and hydrogens that has the desired molecular
mass, M.

 The index of hydrogen deficiency (unsaturation index), U, that corresponds
to the preceding formula is calculated easily by applying the relationship
U = ( n - r + 2 ) /2
[ Table :Carbon/Hydrogen Equivalents for some common elements]
(11) Clinical studies
 In any disease condition, the chemistry of body changes which results in the changes
in products in body fluids and excretion products can be detected for the diagnosis
purpose by chromatographic instrument like gas chromatography equipped with
mass spectroscopy. Matrix-assisted laser desorption/ionization mass spectrometry
(MALDI-MS) is now in trend that is used to directly analyze and image
pharmaceutical compounds in intact tissue.

(12)Pharmaceutical analysis
 Mass spectroscopy emerged as a powerful tool for various operations in
pharmaceutical field mainly in drug development.
 Mass spectroscopy now becomes an irreplaceable tool in all types of drug discoveries
due to its high sensitivity, speed, versatility and selectivity .
(13) Forensic applications
 The sample for forensics in the case of drug abuse is mainly urine, hair and blood.
Some of the drugs in routine analysis include opiates, cocaine, marihuana, lysergic
acid diethylamide (LSD) and amphetamines. However, cases of murders or death
due to poisoning and drug overdose are also the prime targets for these drug
candidates’ analysis .
(14) Metabolites analysis
 Determination of metabolic pathway and different metabolites of a drug or
xenobiotics is very important to assess its different parameters of pharmacokinetics.
 Drug metabolic reactions can be divided into two parts: 1. Phase I or
functionalization reactions and 2. Phase II or conjugation reactions. Both of
these transformations involve changes in the molecular weight. These changes can be
accurately measured by mass spectrometer.
 In structural characterization by mass spectrometry, the exchange of labile hydrogen
with deuterium (H/D exchange) in small organic molecules has been widely used and
this occurs in solution containing functional groups which have labile hydrogen(s)
such as -SH, −OH, −N(R)H, −NH2 and -COOH.

 Mass spectrometry (MS) is a powerful analytical tool with many applications in
pharmaceutical and biomedical field. The increase in sensitivity and resolution of
the instrument has opened new dimensions in analysis of pharmaceuticals and
complex metabolites of biological systems. Compared with other techniques, mass
spectroscopy is the only technique for molecular weight determination,
through which we can predict the molecular formula. It is based on the
conversion of the sample into ionized state, with or without
fragmentation which are then identified by their mass-to-charge ratios
(m/e).
 Mass spectroscopy provides rich elemental information, which is an important
aspect to interpret complex mixture components. Thus, it is an important tool
for structure elucidation of unknown compounds. Mass spectroscopy also
helps in quantitative elemental analysis, that is, the intensity of a mass spectra
signal is directly proportional to the percentage of corresponding element. It is also
a noninvasive tool that permits in vivo studies in humans. Recent research has
looked into the possible applications of mass spectrometers in biomedical field. It is
also used as a sensitive detector for chromatographic techniques like LC–MS, GC–
MS and LC/MS/MS.
5. Conclusion

6. References :
1. R. M. Silverstein, F. X. Webster, and D. Kiemle, Spectrometric Identification of
Organic Compounds, 7th ed., New York: Wiley, 2004; F. McLafferty and F. Turecek,
Interpretation of Mass Spectra, 4th ed., Mill Valley, CA: University Science Books,
1993.
2. McLafferty, F. W., Interpretation of Mass Spectra, University Science Books, Mill
Hill, California (1980).
3. http://webbook.nist.gov/.
4. For recent developments in LC/MS/MS, see R. Thomas, Spectroscopy, 2001, 16, 28,
5. See N. L. Kelleher, Anal. Chem., 2004, 76, 196A; F. W. McLafferty, Int. J. Mass
Spectrom., 2001, 212, 81.
6. See P. M. Peacock and C. N. McEwen, Anal. Chem., 2004, 76, 3417; H. Pasch and W.
Schrepp, MALDI-TOF Mass Spectrometry of Polymers, Berlin: Springer-Verlag,
2003.
7. J. H. Beynon and A. E. Williams, Mass and Abundance Tables for Use in Mass
Spectrometry, New York: Elsevier, 1963.
8.J. C. A. Wuilloud, S. R. Gratz, B. M. Gamble, and K. A. Wolnik, Analyst, 2004, 129,
150: E. W. Taylor, W. Jia, M. Bust G D Dollinger, Anal. Chem., 2002, 74, 3232; L.
Howells and M. J. Sauer, Analyst, 2001, 126, 155,
9. Yinon. Forensic Applications of Mass Spectrometry, Boca Raton, FL: CRC Press,
1995.

10. For reviews of Fourier transform mass spectrometry, see R. M. A. Heeren, A. J.
Kleinnijenhuis, L. A. McDonnell, and T. H. Mize, Anal. Bioanal. Chem., 2004,
378, 1048; A. G. Marshall, Int. J. Mass Spectrom., 2000, 200, 331; A. G. Marshall
and P. B. Grosshans, Anal. Chem., 1991, 63, 215A
11. For a description of commercially available spectrometers designed specifically
for electrospray ionization, see L. Vorees, Anal. Chem., 1994 66, 481A. For
spectrometers combining electrospray with TOF mass analyzers, see C. M. Henry,
Anal. Chem., 1999, 71, 197A.
12. For more information, see J. T. Watson, Introduction to Mass Spectrometry, 3rd
ed., Philadelphia: Lippincott-Raven, 1997, Chap. 9: K. Biemann, Anal. Chem.,
1986, 58, 1288A.
13. Milne,G.W.A. (Ed.), Mass Spectrometry. Techniques and Applications, Wiley,
New York (1972).
14. Chapman, J. R., Practical Organic Mass Spectrometry, Wiley, New York, (1985).
15. Lederburg,J., Computation of Molecular Formulas for Mass Spectrometry,
Holden-Day, San Francisco (1964).
16. Lawson, G. and Todd, J. F. J. Radiofrequency quadrupole mass spectrometers.
Chem. Brit., 7, 373 (1971).
17. http://www.chm.bris.ac.uk/ms/msms.xhtml

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