DRUG DISCOVERY

&
DEVELOPMENT
Principles and Procedures and
Applications of instrumental
methods

Alliance Institute of Advanced Pharmaceutical & Health Sciences

Prafulla Kumar Sahu
M. Pharm (PhD.)
www.allianceinstitute.org
 New drug development
• A process which applies to drugs, products and
protocols to be used on human subjects.

• New Drug development is divided into 4 phases

1.Drug discovery
2.Pre clinical development
3.Clinical trial
4.Post approval

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 Organic Chemistry involved in
Synthesis & Purification
• Organic chemists synthesize new drug compounds as well as
isolate and characterize natural products, such as alkaloids.
• In each case, we have to evaluate the complex relationships
between chemical structure and pharmacological action.
– The pharmacological activity of a compound is an involved function of the
structure, and very small changes may pro-foundly modify the
pharmacological effect (SAR).
Structural modifications:
– replacing one group with another at a specific point in the molecule,
– shifting the same group from place to place in the parent molecule,
– saturating valence bonds,
– modifying the acidity or basicity.
• Chromatographic techniques have been widely used for the
purification of newly synthesized compounds.
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 Instrumental Techniques for
Product Characterization
• The first step in product characterization is to
establish the precise chemical identity of the
product. It is important to determine whether the
material is a compound, i.e.
– a single chemical entity,
– a mixture of closely related compounds,
– mixture of isomers, or
– merely a loose molecular complex of readily dissociable
components.
• Such information is fundamental to a proper
evaluation of the biological properties of the
material. Alliance 4
 Product Characterization
• For compounds of synthetic origin:
• Identity is usually clearly defined by the
synthetic route employed.
• Modern spectroscopic techniques:
– 1H and 13C NMR
– IR (infrared spectroscopy) are sensitive tools for
such purposes.

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 Physicochemical characterization
techniques
• Play a major role in the drug development
process because they help us to understand
the mechanism of drug delivery.
• Dissolution is the first step in drug delivery by
solid oral dosage forms.
– but the determination of in vitro release profiles
provides little information on the mechanism of
the release of API from the product matrix.

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• It is required to assess (using instrumental techniques):
– Internal structure of the dosage form
– Microhomogeneity
– Morphology of the api in the dosage form
• instrumental techniques used:
– Microscopy and energy-dispersive x-ray spectroscopy
– X-ray powder diffraction,
– Thermal analysis,
– FTIR microspectroscopy,
– NMR imaging,
– Mass spectrometry, and
– Raman spectroscopy.
• The collection and interpretation of the results obtained from
these techniques can be used to optimize formulation
development and ensure the consistency, quality, and stability of
solid dosage forms. Alliance 7
 Product purification
• Powerful separative techniques, particularly
chromatography in all its many forms, provide
sensitive methods.
• Liquid chromatography: is used for both purification
and quantitative determination of the composition
of mixtures of related compounds such as mixtures
of isomers.
• Capillary gas chromatography: The speed and high
separative power make it particularly useful, if highly
specialized technique for the separation of complex
mixtures during the research phase of drug
development. Alliance 8
• Where the product consists of more than one isomer, the isomers
must be capable of separate identification and measurement to
establish means of ensuring batch-to-batch consistency of isomer
composition.
• For most optically active compounds, a simple polarimetric
measurement will sufficient.
• Occasionally, however, where the measured rotation is small,
measurements on a more sensitive instrument at other
wavelengths either directly or after derivatisation may be necessary
to secure adequate control of the product.
• In exceptional cases, the identification of optical isomers differing in
only one of several chiral centers may call for the use of optical
rotatory dispersion or circular dichroism to provide a degree of
sensitivity that cannot be obtained from simple measurements of
optical rotation.
• NMR is particularly valuable in distinguishing geometrical isomers.

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 Goals and objectives
• Clinical pharmacology and pharmacometrics
• Safety
• Activity
• Effectiveness
• Differentiation
• Successful FDA submission
• Market expansion and post marketing surveillance

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 Drug discovery process
• Screening consists of testing many compounds
in assays relevant to the disease in
question……HIT
• If the compound or its structural derivatives
continue to show promise after further
biological and chemical characters….LEAD

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Two basic strategy are applied
1. Compound centered
2. Target centered

1. Compound centered approach:

• Natural products: isolated from plants,
animals and microorganisms.
ex. Morphine, Quinine, Atropine
Adrenaline, Thyroxine
Penicillin
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 Natural ligands

Examples:
• Dopamine- Parkinson- L-dopa
• Insulin – diabetes – exogenous insulin

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2. Target centered approach

Use biochemical or molecular target

Adv :
• pharmacological activity
• system assay

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 Structure based approach
• Use 3-D structure of the target obtained
through x-ray crystallography or NMR.
• Adv :
-potency
-limited number of drugs
• Disadv :
-synthesis

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• Chemical synthesis:
Based on SAR - ex. Histamine blockers
Based on enantiomers - ex. dopa
• Rational approach:
ex. Proton Pump Inhibitors.
• Molecular modelling:
ex. COX 2 inhibitors
• Combinatorial chemistry:
• Biotechnology:
ex. Growth factors, cytokines.
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 Lead optimization
• In this stage where physical, chemical, biological and
pharmacological properties are characterized and refined with
the ultimate goal of selecting a single molecule to enter into
clinical testing and formal drug development.
Reasons:
• Failure to demonstrate Efficacy
• Low bioavailability
• Extensive metabolism
• Low solubility
• Toxic effects
• Cost effectiveness in synthesis
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 Drug discovery
• Identifies interesting drug compounds, drug
targets and delivery mechanisms with the
potential for development into products.
• Characterize compounds and their targets
• Are “proof of concept” in nature
• Provide fundamental information on target
biological system
• Identify lead compounds for further
development
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 Pre clinical drug development
• Space the gap between drug discovery and
clinical trials.
• Provide data on the safety and efficacy of the
product
• Includes In vitro study, drug manufacturing,
formulation and packaging, In vivo studies

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 In vitro study
• Intact cell lines
assess drug effects on cells with specific DNA
mutation
• Cell free system
assay of enzyme activity, receptor binding,
protein interaction with signal transduction

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Drug manufacturing and formulation
• Conducted under current good
manufacturing practices (CGMP) guidelines
IN-VIVO STUDIES
• PK study – ADME study
• PD study
• Therapeutic index
• Toxicological study

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 Toxicological study
Objectives :

Acute study

• Method

• Aim : to provide safety/therapeutic index

• Observation

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 Sub acute study
aim :
• To identify the target organ
• To determine the clinical parameters
• To estimate the safety margin
Method:
Evaluation:

Chronic study
Similar to sub acute study except for the
duration

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 Special study

• Effect on reproductive system and

Teratogenicity

• Mutagenicity- ame’s test

• Carcinogenicity

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 Clinical drug evaluation
Authorization
• Investigational new drug (IND) submission
-the rationale for the drug and patient group
to be treated
-all pre clinical safety and efficacy data
-detailed plan for clinical development
-CIB( clinical investigators brochure)
• Submitted to FDA for review and permission to
proceed.

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 Does it
work in
double
Is it safe? Does it work? blind
trials?

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 APPROVAL

• Once all clinical data has been submitted,

reviewed and approval is granted to license
in market
• Post marketing surveillance
• Approval takes 6 months to 2 years

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 Analytical Chemistry
• Definition:
• “The Science & art of determining
composition of material in terms of element
or compound content”
• Concerned with Chemical Characterization of
mater
• Involves Qualitative and Quantitative
determination

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 Goal of Analytical methods

Qualitative Quantitative
analysis Analysis analysis

Qualitative analysis: A type of chemical analysis

by which the analyte or analytes in a sample are
identified.
Quantitative analysis: A type of chemical analysis
by which the amount of each analyte or analytes in
a sample is determined.
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Classification of Analytical Methods

Analytical
Techniques

Chemical methods Physical methods

(Classical methods) (Instrumental methods)

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Chemical Method (Classical Method)
• Gravimetric Method
• Volumetric Method
– Acid Base titrations (Neutralization method)
– Oxidation reduction titrations (Redox method)
– Precipitation titrations
– Complexometric titrations

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 Classical Methods of Analysis
• Early years of chemistry
• Separation of analytes by precipitation,
extraction, or distillation.
• Qualitative analysis by reaction of analytes with
reagents that yielded products that could be
recognized by their colors, boiling or melting
points, solubilities, optical activities, or refractive
indexes.
• Quantitative analysis by gravimetric or by
titrimetric techniques.
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Instrumental (Physical) Methods

Instrumental
Methods

Optical Electrochemical Miscellaneous

methods methods methods

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 Instrumental Methods
• Measurement of physical properties of analytes - such
as conductivity, electrode potential, light absorption or
emission, mass-to-charge ratio, and fluorescence-
began to be employed for quantitative analysis of
inorganic, organic, and biochemical analytes
• Efficient chromatographic separation techniques are
used for the separation of components of complex
mixtures.
• Instrumental Methods of analysis (collective name for
newer methods for separation and determination of
chemical species.)

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 Optical Methods
Optical methods
(Interaction of EMR)

Scattering
Absorption Emissions Rotation of
or Refraction
of EMR of EMR
of EMR EMR

UV-Visible
Nephlometry
IR Fluoroscence Turbidometry
NMR Phosphorescence Raman Spectroscopy
Polarimetry
X-Ray Flame Photometry Refractrometry
Spectroscopy

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 Electro Chemical Methods
• pH metry & Potentiometry
• Conductometry
• Voltametry
• Polarography
• Amperometery
• Coulometry

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 Miscellaneous methods
• Thermometric Analysis:
– DSC
• Thermo Gravimetric Analysis

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 Classification of Analytical Methods
Based on goal of application:

1. Classical methods

Qualitative – identification by color,

indicators, boiling or melting points, odors

Quantitative – mass or volume (e.g.

gravimetric, volumetric)

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 2. Instrumental methods
Qualitative – chromatography, electrophoresis
and identification by measuring physical
property (e.g. spectroscopy, electrode potential)

Quantitative – measuring property and

determining relationship to concentration (e.g.
spectrophotometry, mass spectrometry)

Often, same instrumental method used for

Qualitative and Quantitative analysis.
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 Types of Instrumental Methods

Signal Example Method

Radiation emission Emission spectroscopy
(X-ray, UV, visible),
fluorescence,
phosphorescence,
luminescence

Radiation absorption Absorption spectroscopy

spectrophotometry,
photometry, NMR
electron spin resonance
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 Signal Example Method
Radiation Scattering Raman spectroscopy

Radiation refraction Refractometry

Radiation diffraction X-ray and

Electron diffraction method

Radiation rotation Polarimetry

Electrical potential Potentiometry

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 Signal Example Method
Electrical charge Coulometry

Electrical current Voltammetry – amperometry

polarography

Electrical resistance Conductometry

Mass Gravimetry

Mass-to-charge ratio Mass spectrometry

Rate of reaction Flow injection analysis

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 Chemical and physical properties employed in instrumental methods:

Characteristic Instrumental Methods

property
Emission of radiation Emission spectroscopy (X-ray, UV, visible, electron);
fluorescence, phosphorescence, and luminescence (X-ray, UV,
and visible)
Absorption of radiation Spectrophotometry and photometry (X-ray, UV, visible, IR);
photoacoustic spectroscopy; nuclear magnetic resonance and
electron spin resonance spectroscopy
Scattering of radiation Turbidimetry; nephelometry; Raman spectroscopy
Refraction of radiation Refractometry; interferometry
Diffraction of radiation X-Ray and electron diffraction methods
Rotation of radiation Polarimetry; optical rotary dispersion; circular dichroism
Electrical potential Potentiometry; chronopotentiometry
Electrical charge Coulometry
Electrical current Polarography; amperometry
Electrical resistance Conductometry
Mass-to-charge ratio Mass spectrometry
Rate of reaction Kinetic methods
Thermal properties Thermal conductivity and enthalpy
Radioactivity Activation and isotope dilution methods

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Instruments for Analysis

Analytes
Energy Analytical
Data
stimulus response
(in matrix)
Encoded
information
Example: Spectrophotometry
Instrument: spectrophotometer
Stimulus: monochromatic light energy
Analytical response: photocell
Data: electrical current
Data processor: current meter
Readout: meter scale
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Encoded information:
detector : device that indicates changes in environment
transducer : device that converts non-electrical to
electrical data

Non-electrical domains Electrical domains

Physical Current
(light intensity, colour)

Chemical (pH) Voltage

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Advantages of Instrumental Methods
• High Sensitivity
• Accuracy
• Small Sample can be used
• Measurements obtained are reliable &
Reproducible
• Fast Determination
• Complex sample can be handled easily Process
can be made automatic
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Limitations of Instrumental methods
• High Cost
• Necessary to use reference substance
• Skilled Persons are required

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 Selecting an Analytical Method

Based on answers to the following questions:

1. What accuracy and precision are required?
2. How much sample is available?
3. What is the concentration range of the analyte?
4. What components of the sample will cause
interference?
5. What are the physical and chemical properties of the
sample matrix?
6. How many samples are to be analyzed?

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IR Spectroscopy
 INFRARED REGION
• 4000cm-1 to 600 cm-1
• IR spectrum:
– Plot of Trasmittance/Absorbance VS Frequency of
radiation
SAMPLE
IR TRANSMITTED
ABSORBED

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 Molecular vibration
• Atoms joined by covalent bonds undergo
continual vibrations relative to each other
• The energies associated with these vibrations
are quantitized. Within a molecule only
specific vibrational energy levels are allowed.
• The energies associated with transitions
between vibrational energy levels for most
covalent bonds are from 2 to 10 kcal/mol (8.4
to 42kj/mol)

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 Infrared spectroscopy
Vibrational IR spectral region covers 2.5 x 10-6
(2.5 µm) to 2.5 x 10-5 (25 µm)
Absorption of IR radiation in this region causes
bond to change from a lower vibrational
energy level to a higher one
• IR SPECTRUM REPRESENTS CHANGES IN
ENERGY BROUGHT BY TRANSITION OF
MOLECULES FROM ONE VIBRATIONAL OR
ROTATIONAL ENERGY STATE TO ANOTHER.

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• IR causes a vibration only if there is a change
in dipole during vibration
• Therefore symmetric bonds are inactive.
– CH3 - CH3 will not observe IR stretch for C-C bond
of ethane

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 Infrared (IR) and Raman Spectroscopies
• Infrared (IR) and Raman spectroscopies have been standard
methods of analytical pharmacy and chemistry for a long
time. IR and Raman spectra, which are complementary to
each other, provide images of vibrations of the atoms of a
compound. Therefore, both techniques are also referred to as
vibrational spectroscopy.
 An IR spectrum is obtained by passing infrared radiation through a
sample and determining what fraction of the incident radiation is
absorbed at particular frequency.
 On the other hand, a Raman spectrum is obtained by focusing
monochromatic radiation on a sample and analyzing the scattered
light as a function of frequency.
• IR spectroscopy is based on the absorption of
electromagnetic radiation by a molecular system,
• Raman spectroscopy relies upon inelastic scattering of
electromagnetic radiation by a molecular system.
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 Applications of IR
• Fourier Transformed Infrared (IR) and Raman Spectroscopy
are important tools for the solid state characterization of
pharmaceutical solids and for the identification of their
chemical structures.
• Generally both methods are applied in combination with
other methods for solid state characterization of
pharmaceutical solids (e.g. X- ray powder diffraction, DSC,
TG).
• characterization of polymorphism and detection in drug
product.
• Both methods are also suitable for the identification/
detection of the polymorphic form in the tablet.
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 Applications of FTIR
• Very high resolution required- Gaseous
Mixtures
• Study of samples having very high absorption
• Study of samples with weak absorption bands
• Used in protein structure determination
• Used in characterizing Adrenocarcinoma
• Very small sample size:
– Obtaining Reflection spectra
– IR emission study Alliance 56
 Applications of Near IR
• NIR: good penetration properties
• Minimal sample penetration required
• Thick layers can be analyzed
• Not much useful for identification
• Quantitative analysis of compounds
containing functional groups made of H
bonded to O, C, N
• Determination of 10, 20, 30 amines

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 Applications of ATR-FTIR
• Surface analysis of biological structures
• Monitoring reaction processes
• Evaluation of fermented foods

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 Pharmaceutical applications of
Mid-IR and Raman spectroscopy
• Mid-IR and Raman spectroscopy are versatile tools in
pharmaceutics and biopharmaceutics, with a wide
field of applications ranging from characterization of
drug formulations to elucidation of kinetic
processes in drug delivery.
• New developments in applications of these methods
for studying drug delivery systems.
• FTIR-ATR is a well-established standard method used
to study drug release in semisolid formulations, drug
penetration, and influence of penetration modifiers;
it is also capable of in vivo studies.
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• FTIR-PAS (photo acoustic spectroscopy) has been applied to:
– Measure drug content in semisolid and solid formulations,
– Determine drug penetration into artificial and biological
membranes.
• Raman spectroscopy can be used to:
– Characterize the structure of colloidal drug carrier systems.
– In vivo studies.
• Recently, there has been tremendous technical improvement in
vibrational microspectroscopy:
– FTIR imaging shows great promise in its ability to visualize
the drug and excipient distribution in pharmaceutical
formulations such as tablets and therapeutic transdermal
systems, as well as to reveal the mechanism of drug release.
– Furthermore, this unique technique offers completely new
possibilities to study the lateral diffusion of drugs.
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NUCLEAR MAGNETIC RESONANCE
SPECTROSCOPY

-an ideal tool for Diagnosis &

Drug Design

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 INTRODUCTION
• BASIC PRINCIPLE IN NMR
• Atomic Nuclei – tiny magnets
• In external Magnetic Field - allign or oppose
• If irradiated with EMR of proper frequency – Resonance
√ α βo
PARAMETERS
 Chemical shifts
 Spin lattice relaxation time

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NUCLEAR SPIN STATES - HYDROGEN NUCLEUS
The spin of the positively
m charged nucleus generates
a magnetic moment vector, m.

+ +

The two states

m are equivalent
in energy in the
+ 1/2 - 1/2 absence of a
magnetic or an
TWO SPIN STATES electric field.
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 N
N
N - spin state, S - spin state,
favorable, unfavorable,
lower energy higher energy
S N
S S

A spinning nucleus with it's magnetic field A spinning nucleus with it's magnetic field
aligned with the magnetic field of a magnet aligned against the magnetic field of a magnet

at no magnetic field, proton spin state

there is no difference beteen (higher energy)
- and - states.
Graphical relationship between
magnetic field (Bo) and frequency ( E E = h x 300 MHz E = h x 500 MHz

for 1 H NMR absorptions

proton spin state
(lower energy)

0T 7.05 T 11.75 T

Bo
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 RESONANCE

Absorption of energy by the spinning nucleus

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 Resonance

• Resonance: Matching of natural frequency with

the applied frequency.
• In NMR the applied radio frequency has to be
match with the precessional frequency of
nuclei (which is the consequences of applied
external magnetic field)
• Absorption of radio frequency occur by nuclei
after resonance is achieved.

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 Precessional frequency (n)
The number of revolution per second made by
magnetic moment vector of the nucleus around the external
magnetic field B0
OR

Precessional frequency is equal to the frequency of

electromagnetic radiation in MHz (Mega cycles/sec)
necessary to induce a transition from one spin state to other.

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 Nuclear Spin Energy Levels
N

-1/2

In a strong magnetic
unaligned
field (Bo) the two
spin states differ in
energy.

+1/2

aligned
Bo Alliance
S
68
 Absorption of Energy
quantized
Opposed

-1/2 -1/2

DE

DE = hn
Radiofrequency

+1/2 +1/2
Applied
Field
Aligned
Bo Alliance 69
THE ENERGY SEPARATION DEPENDS ON Bo

- 1/2

DE = kBo = hn
degenerate
at Bo = 0

+ 1/2

Bo
increasing magnetic field strength
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 The Larmor Equation
DE = kBo = hn can be transformed into
gyromagnetic
ratio g
frequency of g
the incoming
radiation that n = Bo
will cause a
transition
2p
strength of the
magnetic field
g is a constant which is different for
each atomic nucleus (H, C, N, etc)
Stronger magnetic fields (Bo) cause
n α Bo the instrument to operate at higher
frequencies (n).
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 NMR Signals

• The number of signals shows how many different

kinds of protons are present.
• The location of the signals shows how shielded or
deshielded the proton is.
• The intensity of the signal shows the number of
protons of that type.
• Signal splitting shows the number of protons on
adjacent atoms.

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 DIFFERENT TECHNIQUES
• Based on nuclei
• H1 NMR
• C13 NMR
• F13 NMR & P31 NMR
• N15 NMR & O17 NMR

 CWNMR (continuous wave NMR)

 FTNMR

 2D_NMR &3D_NMR
 COSY _CORRELATION SPECTROSCOPY
 MRI_MAGNETIC RESONANCE IMAGING
 ESR_ELECTRON SPIN RESONANCE SPECTROSCOPY

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APPLICATIONS OF NMR IN MEDICINE

 CLINICAL APPLICATION OF PROTON IMAGING IN

DIAGNOSIS
BRAIN
 Distinguishing gray matter & white matter
 Imaging posterior fossae, brain stem, spinal cord
 Detect demyelinating lesions, tumors, hemorrhages,
infarctions
ABDOMEN
o Metabolic liver disease
o Measures liver iron over load in hemochromatosis
o Focal areas of inflammation in chronic active hepatisis
KIDNEYS
o Distinguishing renal cortex & medulla
o To evaluate transplanted kidney
PELVIS
o Differentiates between Benign prostatic hyperplasia
& prostatic carcinoma
o Detects bladder tumours

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LUNG
o Assessing abnormalities
o Respiratory gating

HEART
o Tomographic images of heart muscle, chambers,
valvular structures
o ECHO-PLANAR TECHNIQUE
o Discrimination between infarcted, ischemic & normal
myocardium

BREAST
o 3D-NMR & single-slice planar imaging in detecting
breast abnormalities

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MUSCULO SKELETAL SYSTEM
 Demonstrates Osteo myelitis, tumor metastasis in
vertebral bodies & pelvic bones
 Images of muscles, tendons, ligaments

BLOOD VESSELS & FLOW

 Atherosclerotic vascular disease
 Assess blood flow in major vessels.

INVIVO SPECTROSCOPY
 Chemical shift phenomenon
 Diagnosis of rare disease to inborn errors- 
Mc Ardle’s syndrome
 INVIVO Analysis of Bone flouride content.
 NMR IMAGING STUDIES

 Eliminates risk of x-radiation

 Excellent spatial & contrast resolution

 Detecting diseases at earlier stages

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 NMR IN PHARMACEUTICAL RESEARCH
 Leading technology for 3-D structure determination
of bio-macromolecules
 Studying protein structure
 SAR by NMR – Novel lead compounds
 Chemical shift mapping – Structural information on
the binding modes and site positions
 Molecular dynamics, conformational analysis
 SHAPES – Lead generation
 NMR – SOLVE

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Structural genomics
Identifying gene products in disease
Targets of drug design.

Development of Novel drug delivery systems

Enhancing HTS assays

Thousands of compounds
Cryogenic NMR technology / cryoprobes

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1.Identification of structural isomers
2.Detection of hydrogen bonding
3.Detection of aromaticity
4.Distinction between cis and trans isomer
5.Detection of electronegative atom/group
6.Detection of some double bond character due to
resonance
7.Importance in quantitative analysis

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 NMR Nobel Prize Laureates
• The Nobel Prize in Physics 1943, Ohostern, USA –
Discovery of magnetic moment of proton
• The Nobel Prize in Physics 1952- IsidorJ. Rafi,USA –
Discovery of new methods for nuclear magnetic
precision
• The Nobel Prize in Chemistry 1991 – Felix Bloch, USA
and Edward M. Purcell, USA – Methodology of high
resolution NMR.
• The Nobel Prize in Chemistry 2002 – Kurt Wuthrich,
Switzerland – Determining 3-D Structure by NMR.
• The Nobel Prize in Medicine 2003 – Paul C. Lauterbur,
USA and Peter Mans field, U.K. – Discoveries
concerning MRI.
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MASS SPECTROMETRY

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 Introduction
• Mass spectrometry is employed to analyze
combinatorial libraries, sequence biomolecules,
• Structure elucidation of unknowns,
• Environmental and forensic analytics,
• Quality control of drugs, flavors and polymers

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• The basic principle of mass spectrometry (MS):
– To generate ions from either inorganic or organic
compounds by any suitable method,
– To separate these ions by their mass-to-charge ratio (m/z)
– And to detect them qualitatively and quantitatively by
their respective m/z and abundance.
– Besides electrons, (atomic) ions or photons, energetic
neutral atoms and heavy cluster ions can also be used to
effect ionization of the analyte.
– The time-of-flight (TOF) analyzer, ion separation by m/z
can be effected in field free regions, too, provided the ions
possess a well defined kinetic energy at the entrance of
the flight path.
The analyte may be ionized thermally, by electric fields or
by impacting energetic electrons, ions or photons.
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 Application of LDI (Laser Desorption/Ionization)
• LDI of peptides and oligosaccharides
• LDI is much better suited for the analysis of organic and inorganic
salts
• Molecules with large conjugated π-electron systems
• Organic dyes as contained in ball point inks
• Porphyrins
• UV light-absorbing synthetic polymers
• LDI presents a useful alternative to MALDI in the low-mass range.
• In addition, solvent free sample preparation can be employed
with insoluble analytes.
• However, LDI is a “harder” method than MALDI and
fragmentation

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 Applications of MALDI
1. MALDI-MS of Synthetic Polymers:
2. Fingerprints by MALDI-MS
3. Carbohydrates by MALDI-MS
4. Structure Elucidation of Carbohydrates by MALDI
5. Oligonucleotides in MALDI

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• MALDI is the method of choice for the analysis of synthetic polymers
because it usually provides solely intact and singly charged
quasimolecular ions over an essentially unlimited mass range.
• While polar polymers such as poly (methylmethacrylate) (PMMA),
polyethylene glycol (PEG), and others readily form [M+H]+ or
[M+alkali]+ ions,
• Nonpolar polymers like polystyrene (PS) non-functionalized
polymers like polyethylene (PE) can only be cationized by transition
metal ions in their 1+ oxidation state.
• The formation of evenly spaced oligomer ion series can also be
employed to establish an internal mass calibration of a spectrum.
• The most important parameters that can be determined by MALDI
are
– number-average molecular weight (Mn)
– weight-average molecular weight (Mw)
– molecular weight distribution expressed as polydispersity (PD)
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 Application of Electrospray Ionization
• ESI of Small Molecules (polar analytes)
• Peptide and protein characterization including their complete
sequencing
• ESI is applied to Metal Complexes (ionic) and related
compounds
• ESI of Surfactants: Cationic, anionic and non-ionic surfactants
are readily detected by ESI
• Oligonucleotides, DNA, and RNA are best analyzed by
negative-ion ESI
• ESI of Oligosaccharides and closely related compounds such
as glycoproteins, gangliosides, liposaccharides etc. permits
molecular weight determination and structure elucidation

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LC-MS Applications in
drug development

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 LC-MS Applications in drug development
Development Stage LC/MS analysis activities
Drug discovery Protein identification; natural products
identification; metabolic stability profiles;
molecular weight determination for
combinatorial/medicinal chemistry support
Preclinical Impurity, degradant, and metabolite
development identification
Clinical Quantitative bioanalysis; structure identification
development
Manufacturing Impurity and degradant identification

Alliance 91
LC-MS Applications in
Drug Discovery

Alliance 92
 Peptide Mapping
1. Peptide Mapping:
• The identification of proteins is essential for understanding
biological process and the function of the protein during
normal and disease states.
• The resulting insights lead to the development of therapies
for intervention, and ultimately, the cure of disease. The
information and knowledge derived from this type of study
are extremely valuable for activities involved with target
identification activities during drug discovery.
• Two-dimensional gel electrophoresis (2-DGE) is the primary
analysis tool used in the pharmaceutical industry to
characterize the expression of proteins. Mass spectrometry-
based methods are emerging as an important partner with 2-
DGE for correlating proteins to their function.
Alliance 93
 Peptide Mapping
• Identification schemes that involve LC/MS and
MALDI-TOF techniques have been applied to a broad
range of events that involve proteins such as
covalent modification, proteolytic cleavage, and
binding.
• Usually, the focus of LC/MS methods for protein
characterization is on four distinct goals:
– (1) confirmation of putative sequence;
– (2) identification of amino acid modifications;
– (3) identification of known proteins;
– (4) sequence determination of unknown proteins.

Alliance 94
 Glycoprotein Mapping
• Similar analysis strategies can be applied for the
peptide mapping of glycoproteins combined in-
source collisionally induced dissociation (CID) with
LC/MS/MS to identify sites of N- and O-linked
glycosylation. This novel approach uses a series of
LC/MS and LC/MS/MS experiments to generate
peptide maps and to selectively screen for
glycoproteins.
• The method is based on the characteristic
fragmentation of glycoproteins to form a N-
acetylhexosamine (HexNAc+) fragment ion at m/z
204. This fragment ion serves as a diagnostic marker
for N- and O-linked glycopeptides.
Alliance 95
• A mixture of peptides and glycopeptides is generated when
the glycoprotein is reduced, alkylated, and enzymatically
digested.
• A series of three separate experiments, is used to identify the
N- and O-linked glycopeptides.
1. The first experiment uses LC/MS to provide a map of the
peptide portion of the protein and to indicate the presence
of glycopeptides.
2. The second experiment involves the use of LC/MS/MS to
screen the mixture for compounds that fragment to yield the
diagnostic m/z 204 (HexNAc+). N- and O-linked glycopeptides
are both identified.
3. A final experiment is performed on a sample treated with
peptide N:glycosidase F (PNGase F) to release the N-linked
oligosaccharides and to identify the O-linked glycopeptides
exclusively. Alliance 96
Alliance 97
• Additional structural analysis of glycoproteins
generally requires fragmentation by chemical
or enzymatic cleavage and by separation or
isolation. On-line LC/MS methods provide an
integrated approach for this type of analysis.

Alliance 98
 Natural Products Dereplication
• Natural products offer a source of unique chemical diversity
for the pharmaceutical industry.
• Drugs derived from natural products have been introduced for
the treatment of cancer, immunosuppression, cardiovascular
therapy, and anti-infective therapy.
• Most antibiotics come from secondary metabolites of soil
microorganisms that inhibit bacteria or fungi. Large scale
screening of microorganism fermentations followed by
isolation and structure elucidation is required.
• Because many natural products have been previously
identified, approaches that avoid time-consuming isolation
and provide quick elucidation are essential.

Alliance 99
• a new strategy was introduced by Ackermann et al. that uses
an on-line ESI±LC/MS approach that integrates multi-
component identification, fraction collection, and sample
preparation for bioactivity screening (1996).
• Using this LC/MS based approach, crude extracts are screened
without extensive purification and chemical analysis.
• Less material is required due to the sensitivity of the
technique and chromatographic resolution is retained.
• This resolution would ordinarily be relatively poor with a
fraction collection process. Furthermore, because the ESI acts
as a concentration- dependent detector, the HPLC effluent can
be split between mass spectrometry analysis and fraction
collection. Thus, molecular weight information is obtained,
and approximately 90% of the fractionated material is
recovered for biological testing.
Alliance 100
Alliance 101
 Bioaffinity Screening
• Combinatorial chemistry has changed the strategy of drug
candidate synthesis. As a result, hundreds of thousands of
compounds are now screened against a particular biological target.
Once activity is determined for a mixture, the identification of the
active component(s) is necessary. However, this approach requires
a considerable amount of resources.
• To reduce the time and resources required for screening the large
number of compounds produced by combinatorial chemistry,
approaches featuring the parallel screening of mixtures of
compounds (20±30) have been investigated. The recent studies
performed by Anderegg and coworkers describe the use of
bioaffinity selection LC/MS methods for the identification of active
mixture component(s) (Davis et al., in press). This approach features
an integrated bioaffinity based LC/MS screening method to
separate and identify Lead compounds from mixtures.

Alliance 102
Alliance 103
 In Vivo Drug Screening
• Technology for automated highthroughput bioanalysis.
focused on methods for determining multicomponent
mixtures of drug candidates that involve pharmacokinetics
and metabolic stability.
• These activities were performed during the preclinical and
clinical stages of drug development.
• The need to provide an early assessment of these properties
resulted in new strategies for in vivo drug screening during
the drug discovery stage.
• LC/MS/MS approaches, using relatively simple isolation and
chromatography conditions, present an effective approach to
evaluate new lead compounds for subsequent development
or selection of an optimal drug candidate within a specific
therapeutic area or a targeted class of compounds.
Alliance 104
 Metabolic Stability Screening
• Recently, the use of fast gradient elution LC/MS techniques
was described for high throughput metabolic stability
screening (Ackermann et al., 1998). The method uses a HPLC
column-switching apparatus to desalt and analyze lead
candidates incubated with human liver microsomes.
• The rapid structure identification of metabolites using
LC/MS/MS with an ion trap mass spectrometer (Lopez et al.,
1998).
• In this study, metabolic stability analysis is automated to
provide maximum structural information in combination with
predictive strategies for biotransformation.

Alliance 105
• This feature is unique and avoids the multiple (2±4)
injections that are necessary with other MS/MS
configurations (e.g., tandem quadrupole).
• Along with the significant savings in time, detailed structure
information is generated, which enables a comprehensive
analysis of substructure relationship to be constructed for
each metabolite.
• These automated studies provide unique advantages during
drug discovery, and provide an early perspective on the
metabolically labile sites, or ``soft spots'' of a drug candidate.
This knowledge is useful during lead optimization activities,
and can lead to the initiation of proactive research efforts that
deal with metabolism-guided structural modification and
toxicity.

Alliance 106
 Applications of LC/MS
in Preclinical Development.

Alliance 107
Application of LC/MS in
Clinical Development

Alliance 108
Applications of LC/MS in
Manufacturing

Alliance 109
 Applications of LC-MS
1. Toxicological studies, finding drugs and toxins in
variety of materials with smaller sample volume
2. Urine analysis for toxicology
3. Dope test: doping agents and drugs of abuse
(steroids, diuretics)
4. Impurity profiling
5. Metabolite studies
6. Bio-equivalence and Bioavailability studies
7. Molecular weight
8. Assay of drugs, intermediates
Alliance
etc. 110