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TRANSCRIPT
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Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management
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Introduction
Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management 1 | P a g e
Introduction
Analysis of drug product or pharmaceutical product is important as it concerned with
life. Quality must be built in from the initial stage, to the time it is finally made and sent out.Analytical chemistry is mainly concerned about determining the qualitative and quantitative
composition of material under study. Analysis of pharmaceutical products or of specific
ingredients within the product is necessary to ensure its safety and efficacy throughout all
phases of its shelf life..
Pharmaceutical analysis involves separations, identification and determination of the
relative amount of the component in a sample material. Analytical monitoring of
pharmaceutical product or of specific ingredients within product is required to ensure safetyand efficacy throughout shelf life, including storage, distribution and use.
To determine the drug problems satisfactory it is necessary to identify the conspiracy
which could done by characterization and impurity profiling of the drug. Impurity profiling
helps in accepting or rejecting the API batch. Organic impurities can arise during the
formulation process and storage of the drug substances and the criteria for their acceptance up
to certain limits are based on pharmaceutical studies or known safety aspects. According to
regulatory guidelines, the pharmaceutical studies using a sample of the isolated impurities
can be considered for safety assessment. It is, so, essential to isolate and characterize
unidentified impurities present in the drug sample.
Introduction to Analytical Methods
There are various methods of analysis can be broadly classified into two categories; Classicalmethods and Instrumental methods
Classical Methods (1)
1. Volumetric method: It is based on the determination of a solution of known strengthrequired to complete a chemical reaction with the substance under analysis
2. Gravimetric method: In this method of analysis, the assay results generally obtainedeither by determining the weight of a substance in the sample, or the weight of some
other substance derived from the sample, the equivalent weight of which givess as the
basis for calculation
.
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Instrumental Methods Of Chemical Analysis (2)(3)
Instrumental methods are exciting and fascinating part of chemical analysis that
interacts with all areas of chemistry and with many other areas of pure and applied sciences.
Analytical Instruments plays an important role in the formulation and analysis of newproducts. This instrumentation provides lower detection limits (LOD) required to assure safe
foods, drugs, water and air. Instrumental methods are widely used by Analytical scientists to
utilize time smartly, to avoid chemical separation and to obtain highest possible accuracy.
Most instrumental techniques fit into one of the principal areas like spectroscopy,
electrochemistry, chromatography and miscellaneous techniques.
Most instrumental techniques are based one of the four-principle areas
Spectrophotometric techniques (1):
UV and Visible Spectrophotometry
Fluorescence and Phosphorescence Spectrophotometry
Atomic Spectrophotometry (emission &absorption)
Infrared Spectrophotometry
Raman Spectrophotometry
X-Ray SpectrophotometryNuclear Magnetic Resonance Spectroscopy
Mass Spectroscopy
Electron Spin Resonance Spectroscopy
Electrochemical Techniques
Potentiometry
Voltametry
Electrogravimetry
Conductometry
Amperomertry
Chromatographic Techniques
High Performance Liquid Chromatography (HPLC)
Gas chromatography (GC)
High Performance Thin Layer Chromatography (HPTLC)
Paper chromatography
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Introduction
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Hyphenated Methods
GC- MS (Gas chromatography - Mass Spectroscopy)
LC-MS (Liquid Chromatography - Mass Spectroscopy)
GC- IR (Gas chromatography - Infrared Spectrophotometry)
LC-IR (Liquid Chromatography - Infrared Spectrophotometry)
Miscellaneous Techniques
Thermal analysis
Kinetic Techniques
Electrophoresis
Introduction to Chromatography
Chromatography is unique in the history of analytical methodology and is probably
the most powerful and technique available in the modern analysis. it can able to separate a
mixture into its individual components simultaneously and determine quantitatively the
amount of each component present (4).
Principle (2)
Chromatography is a non-destructive procedure for resolving multi-component
mixture of trace, minor and major constituents into its individual fractions. Chromatography
is primarily a separation tool. The technique of chromatography is based on the difference in
the rate at which components of a mixture move through a stationary phase under the effect
of some solvent or gas (mobile phase). Between the two phases of this system, phase
equilibrium is obtained for all the components of the mixture. The separation may be
successful only if the equilibrium constants of all these components have reasonable value. If
they are too small (too small path length), then compounds travel with almost equal velocity
to that of a solvent and their complete separation could not achieve. If the constants are too
large, then they cannot leave the column. Temperature, the nature of the solid surface and the
nature and composition of mobile phase, combinable affects the equilibrium constant. If solid
phase is an adsorbent, its specific surface area and pore volume are critical factor. The fluid
used as the mobile phase may be liquid, gas or a super-critical liquid. Thus three possible
types of chromatography are liquid chromatography, gas chromatography and super-critical
chromatography(2).
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Figure 1- Retention Process in Chromatography
High Performance Liquid Chromatography (2) (4) (5)
In HPLC, for separation of individual components, the sample is being introducedinto flowing stream of mobile phase which is liquid in case of HPLC, and the analytes are
allowed to pass through a column layer of packing material of small diameters (so large
surface area can be obtained), called stationary phase. As the analyte molecules pass through
the column, along with the moving mobile phase, there is continuous interaction of the
analyte molecules with the stationary phases as well as with the mobile phase. This process is
finally results in a dynamic equilibrium. The differences in the equilibrium processes of the
different solute molecules results in the separation of components from the mixture.
Liquid Partition Chromatography are of two types(1)
Normal Phase Chromatography Reversd Phase Chromatography
1. Normal Phase- The stationary phase is polar and mobile phase is non-polar. In this case,
solute elution is based on the principle that non-polar solute prefer mobile phase and elute
earlier and polar solute prefer the stationary phase and elute later.
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2. Reverse Phase- The stationary phase is non-polar and mobile phase is polar. The solute
elution is reversing of that of normal phase i.e. polar elute earlier as compared to non-polar
which elute later.
Figure 2- HPLC System
Components of HPLC(2)
Typical HPLC system consists of the following main components:
A) Solvent Reservoirs
This provides storage of sufficient amount of HPLC solvents for continuous operationof the system which is equipped with an online degasser system and special filters to isolate
the solvent from the influence of the environment.
B) Pump
This provides the constant and continuous flow of the mobile phase through the
system.
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C) Injector
This allows injection of the analytes mixture into the stream of the mobile phase
before it enters the column; most modern injectors are auto samplers, which allow
programmed injections of different volumes of samples that are withdrawn from the vials inthe auto sampler tray.
D) Column
This is the main part of HPLC system; it actually produces a separation of the
analytes from the mixture. A column is the place where the mobile phase is in contact with
the stationary phase. Most of the chromatography development in now a days went toward
the design of many different ways to increase this interfacial contact.
E) Detector
This is a device for continuous recording of specific physical properties of the
column effluent. The most common detector used in pharmaceutical analysis are UV
detectors allows monitoring and continuous recording of the UV absorbance at a selected
wavelength or over a span of wavelengths (DAD). Flow of the analyte in the detector flow
cell causes the change of the absorbance. If the analyte absorbs greater than the background(mobile phase), a positive signal is obtained.
F) Data Acquisition and Control System
Computer-based system that controls all parameters of HPLC instrument (eluent
composition (mixing of different solvents); temperature, injection sequence, etc.) and
acquires data from the detector and monitors system performance.
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Normal Phase Chromatography Reversed Phase Chromatography
Mechanism
Retention by interaction of the
stationary phases polar surface
with polar parts of the sample
molecules.
Retention by interaction of the
stationary phases non-polar
hydrocarbon chain with non-polar
parts of sample
Stationary
Phase
bonded siloxane with polarfunctional group like SiO2, Al2O3, -
NH2, -CN, -NO2, - Diol
bonded siloxane with non-polarfunctional groups like n- octadecyl
(C-18) or n- octyl (C-8), ethyl,
phenyl, -(CH2) n-diol, (CH2) n-CN
molecules.
Mobile PhaseNonpolar solvents like heptane,hexane, cyclohexane, chloroform,
ethyl ether, dioxane
Polar solvents like methanol,acetonitrile, water or buffer
(Sometimes with additives of THF
or dioxane).
Elution Order Least polar components are eluted first Most polar components are elutedfirst
Table 1- Types of Separations in liquid Chromatography (4) (2)
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Ultra violet spectroscopy(3)
(1)
The wavelength range of UV radiation starts at blue end of the visible light and ends at
2000.
The ultraviolet region is subdivided into two spectral regions:
Wavelength Region
2000 Near UV region
2000- 4000 Vacuum UV region
Ultraviolet absorption spectra arise from transition of electron or electrons within a
molecule or an ion from a lower to a higher electronic energy level (ground state to excited
state) and ultraviolet emission spectra arise from the reverse type of transition (excited state
to ground state). For radiation to cause electronic excitation, it must be in the UV region of
the electromagnetic spectrum.
Theory of spectrophotometry: (2)
Lamberts law:
This law can be stated as follows:
When a beam of light is allowed to pass through a transparent medium, the rate of decreasing
of intensity with the thickness of medium is directly proportional to the intensity of light.
T = I / Io
A = -log T = - log (I / Io)
Beers law
This law can be stated as follows:
When a beam of light is allowed to pass through a transparent medium, the rate of increasing
of concentration is directly proportional to the intensity of light.
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T = C / Co
A = -log T = - log (C / Co)
Beers and Lamberts law
This combine law shows that there exists a logarithmic relationship between the
transmittance and the length of the optical path through the sample. And similar relationship
holds between transmittance and the concentration of the solution that means the intensity of
a beam of monochromatic light decreases exponentially with the increase in concentration of
the absorbing substance arithmetically.
X-ray difractrometer(6)(2)
About 95% of all solid materials can be classified as crystalline. When X-rays interact
with a crystalline substance, a unique diffraction pattern is obtained. The X-ray diffraction
pattern of a pure substance is, so, like a fingerprint of the substance. The powder diffraction
method is thus ideally suited for characterization and identification of polycrystalline
substances..
Solid mattercan be described as:
Amorphous: The atoms are arranged in a random way similar to the arrangement disorder
found in a liquid. Glasses are amorphous materials.
Crystalline: The atoms are arranged in a regular pattern, and there is as smallest volume
element that by repetition in three dimensions (X, Y, Z axis) describes as the crystal.
X-rays can be produced by the bombardment of a target with stream of high energy
particles such as 20 to 50 KeV electrons or nuclear particles from a radioactive source such as
Cu. A typical X-ray generator uses an evacuated tube into which the target projects as a
cooled anode together with a tungsten filament as a cathode. The impact of the bombarding
particles on the target is non-selective and produces a wide range of energy transitions and
continuously emits the of X-ray.
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Introduction
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Figure 3- X-ray diffraction Technique (6)
Differential Scanning calorimeter (DSC)
The basic principle underlying this technique is that when the material undergoes a physical
transformation such as phase transition, more or less heat will need to flow to it than the
reference to maintain both at the same temperature. In that case more or less heat must flow
to the material depends on whether the process is exothermic or endothermic. For example, as
a solid sample melts to a liquid it requires more heat flow to the sample to increase its
temperature at the same rate as the reference. This is because of the absorption of heat by the
sample as it undergoes the endothermic phase transition from solid to liquid. Likewise, as the
material undergoes exothermic processes less heat is required to raise the sample
temperature. By detecting the difference in heat flow between the sample and reference,
differential scanning calorimeter are able to measure the amount of heat absorbed or released
during such transitions. DSC may also be used to observe more subtle physical changes, such
as glass transitions. It is widely used in pharmaceutical industries as a quality control tool due
to its applicability in evaluating sample purity and for studying polymer curing.
Instrumentation
In DSC, a sample and a reference is placed in the instrument. Heaters are either ramp the
temperature at specified rate (10C/min or 5C/min or any other) and the instrument records
the difference in the heat flow between the sample and the reference. The plotted graphobtained from the DSC is called the Thermogram. Thermogram usually shows various
http://en.wikipedia.org/wiki/Meltinghttp://en.wikipedia.org/wiki/Phase_transitionhttp://en.wikipedia.org/wiki/Glass_transitionhttp://en.wikipedia.org/wiki/Glass_transitionhttp://en.wikipedia.org/wiki/Phase_transitionhttp://en.wikipedia.org/wiki/Melting -
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phases of thermal reaction like endothermic or exothermic reaction. With the endothermic
reaction it shows negative peak and with the exothermic reaction it shows positive peak.
Figure 4- DSC Principle
Figure 5- DSC Model thermogram
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Infrared spectroscopy(7) (8)
The infrared region of the electromagnetic spectrum extends from 800 nm to 1mm
and is subdivided into far infrared, near infrared, and very near infrared. The fundamental
region between 2 and 15m is the region that provides the greatest information for theelucidation of molecular functional groups. Particular groups in the molecule, e.g. Hydroxyl,
carbonyl and amines also have characteristic absorption frequencies known as group
frequencies, which are almost independent of the nature of the rest of the molecule.
Region Wavelength
Photographic region Visible to 1.2
Very near IR region
(overtone region)
1.2 to 2.5
Near IR region
(vibration region)
2.5 to 25
Far IR region
(rotation region)
25 to 300
Table 2- IR regions (2)
Modes of vibrations (1)
In a polyatomic molecule, each atom is having three degree of freedom in three direction
which are perpendicular to each other. So polyatomic molecule requires three times as many
degree of freedom as the number of its atom. Thus a molecule of n atoms has 3n degree of
freedom.
Non- linear molecule- it has 3n-6 vibrational degree of freedom.
Linear molecule- it has 3n-5 vibrational degree of freedom.
Normal vibrations are divided into two parts
1. Stretching vibrations2. Bending vibrations
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Stretching vibrations
The atoms move essentially along the bond axis. This vibrations corresponding to the
one dimensional motion, so there will be n-1 stretching vibrationsfor non cyclic systems.
Bending vibrations
In this type, there occurs a change in bond angles between bond with a common atom or there
occurs a movement of group of atoms with respect to the remainder of the molecule without
movement of the atoms in groupwith respect to one another.
E,g. twisting, rocking, torsional
Types of stretching and bending vibrations (7)
Stretching vibrations:
1. Symmetric2. Asymmetric
Symmetric Asymmetric
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Bending vibrations:
1. Scissoring2. Rocking3. Twisting4. Wagging
Scissoring Wagging
Rocking Twisting
Instrumentation
Usually optical materials, glass or quartz absorb strongly in the IR region.
The main parts of the IR spectrometer are as follow
1. IR radiation sources2. Monochromators3. Sample cells4. Detectors
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IR radiation sources (2)
Figure 6- IR sources
Monochromators
The radiation sources emits the radiation of various frequencies. As the sample in IR
spectroscopy absorbs only at certain frequencies, it therefore becomes necessary to select
desired frequencies from the radiation sources and reject the radiation of other frequencies.
This selections has been achieved by means of monochromators, which are major two types.
1. Prism Monochromators2. Grating Monochromators
Sample cells
As IR spectroscopy has been used for the characterization of solid, liquid or gas
samples. It is evident that samples of different phases have to treat differently. But the
common point to the sampling of different phases is that the material containing the sample
must be transparent to the radiation like NaCl or KBr.
IR
radiation
sources
Glober
sources
Mercuryarc
Nnernstglower
Incandescent lamp
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Detectors
Except in the near IR, where a photoconductivity cell is generally used. There is no
better choice than thermal detectors. As the radiant power is low for the IR region, it means
that the detectors signal will also be low. In order to locate this low signals, a preamplifier isfixed to the detector and radiation beam is modulated with a low frequencies light interrupter.
Thus to detect such signals, thermal detectors must possess a short response time and the
absorbed heat must be lost rapidly. The latter condition is most difficult requirement because
heat transfer is not a quick process.
The various types of detectors used in IR spectroscopy are
Figure 7- IR Detectors
Golay cells Photoconductivity cells
Bolometers Thermal detectors
IR Detectors
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Interpretation of IR spectra (7)
There is no rigid rule for the interpretation of the IR spectra. Certain requirement however,must be met before starting of interpretation of IR spectra.
1. The spectrum must be adequately resolved and of adequately intensity.2. The spectrum should be of pure compound.3. Proper calibration should be made with reliable standards such as polystyrene film
Figure 8- simplified chart of common functional group with characteristic absorptions
Bond Mode Wavenumber (cm-1
)
C-H Stretch
Stretch (2v)
Stretch (3v)
Stretch (C)
Bend in plane
Bend out of plane
Rocking
2700 - 3300
56006300
83009000
42005000
13001500
800830
600900
C-C Stretch 800 - 1200
C-O Stretch 900 - 1300
C-C Stretch 8001200
16001700
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Bond Mode Wavenumber (cm-1
)
C=O Stretch
Stretch (2v)
Stretch (3v)
16001900
33003600
50005300
C=N Stretch 16001700
C=C Stretch 21002400
C=N Stretch 21002400
C-F Stretch 10001400
C-Cl Stretch 600800C-Br Stretch 500600
C-I Stretch 500
O-H Stretch
Stretch (2v)
30003700
67007100
N-H Stretch
Stretch (2v)
Stretch (3v)
Stretch (C)
Bending
rocking
30003700
6300- 7100
900010000
48005300
15001700
700900
Table 3- IR positions of various bond vibrations (7)
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Mass spectrometry (2) (8)
Mass spectrometry is the analytical technique in which mixture of gaseous ions were
separated according to their mass-charge (m/z) ratios. A mass spectrum is a plot of relative
pressure or concentration of the gaseous components as a function of the mass-charge.
Mass spectrometry is capable of providing information about:
The element composition of the sample of matter. The qualitative and quantitative composition of complex mixtures, The structure and composition of solid surfaces,
Instrumentation
Figure 9- Mass Spectrometry Instrumentation
Sample inlet systems:
The purpose of the inlet system is to inject of a sample into the ion source with minimal loss
of vacuum. Several types of inlets are:
Batch inlet systems, Direct probe methods, Capillary electrophoretic inlet systems.
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Ionic sources
The function of the ionic sources is to convert the gaseous sample molecules to ions which
can be separated in the mass analyzer based on their m/z, because the energy that is required
for conversion significantly differ the molecules.
The major types of ionic methods of ionization are
Electron- bombardment ionization, Arc and spark ionization, Photo ionization, Thermal ionization, Chemical ionization.
Mass analyzer (1)
Several devices are available for separating ions with different mass to charge ratios. Ideally,
the mass analyzer should be able to distinguishing minute mass differences. Mass analyzer
allows passage of a sufficient number of ions to yield readily measurable ion currents.
Magnetic sector analyzer, Quadrupole mass spectrometers, TOF (time of flight) mass analyzers, Ion trap analyzers.
Mass spectrometry is widely used for the characterization and analysis of high molecular
mass polymeric materials.
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Nuclear Magnetic Resonance (NMR)(9)
(7)
(10)
Nuclei have positive (+ve) charges; many nuclei behave as when they were spinning.
Anything that is charged and moves has a magnetic moment and generates a magnetic field.
so, a spinning nucleus acts as a tiny bar magnet oriented along the spin rotation axis. Thistiny magnet is often called a nuclear spin. If this small magnet when puts in the field of a
much larger magnet, its orientation will no longer be random but it is organized. There will
be one most probable orientation. However, if the tiny magnet is oriented precisely 180 in
the opposite direction, that position could also be maintained. In scientific way, the most
favourable orientation is of the low-energy state and the less favourable orientation is of the
high-energy state. This two-state description is appropriate for most nuclei of biologic
interest including1
H,13
C,15
N,19
F, and31
P; so all those which have nuclear spin quantumnumber I = l/2. It is a main quantum mechanical requirement that any individual nuclear
spins of a nucleus with I = l/2 be in one of the two states whenever the nuclei are in a
magnetic field. It is important to note that the most common isotopes of carbon, nitrogen and
oxygen (12C, 14N and 16O) do not have a nuclear spin because of their spin quantum no is
zero.
Figure 10-The charged nucleus creates a magnetic field B and is equivalent to a small bar magnet whose axis is
coincident with the spin
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The resonance phenomenon
The small nuclear magnet may spontaneously "flip'' from one orientation (energy state) to the
other as the nucleus sits in the large magnetic field. This relatively infrequent event is
illustrated at the left of Figure 10. However, if energy equal to the difference in energies (DE)of the two nuclear spin orientations is applied to the nucleus (or more realistically, group of
nuclei), much more flipping between energy levels is induced (Figure 10). The irradiation
energy is in the RF range (just like on your FM radio station) and is typically applied as a
short (e.g., many microseconds) pulse. The absorption of energy by the nuclear spins causes
transitions from higher to lower energy as well as from lower to higher energy. This two-way
flipping is a hallmark of the resonance process. The energy absorbed by the nuclear spins
induces a voltage that can be detected by a suitably tuned coil of wire, amplified, and thesignal displayed as afree induction decay(FID). Relaxation processes (vide infra) eventually
return the spin system to thermal equilibrium, which occurs in the absence of any further
perturbing RF pulses. The energy required to induce flipping and obtain an NMR signal is
just the energy difference between the two nuclear orientations and is shown in Figure 11 to
depend on the strength of the magnetic field Bo in which the nucleus is placed
where h is Planck's constant (6.63 x 10-27 erg sec). The Bohr condition (DE = hn) enables
the frequency no of the nuclear transition to be written as
Equation is often referred to as the Larmor equation, and wo = 2pno is the angular Larmor
resonance frequency. The gyromagnetic ratio g is a constant for any particular type of
nucleus and is directly proportional to the strength of the tiny nuclear magnet. Table 1.1 liststhe gyromagnetic ratios for several nuclei of biologic interest. At magnetic field strengths
used in NMR experiments the frequencies
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Figure 11- For nuclei (I=1/2) in a magnetic field of strength Bo at thermal equilibrium
Unperturbed, there will be infrequent flips of individual nuclear spins between the two
different energy levels. When a radiofrequency (RF) pulse with appropriate energy is applied
(i.e., equal to the difference in energies of the two levels), transitions between the two energy
levels will be induced, i.e., the nuclear spin system will "resonate"; the spin system absorbs
the energy. Following the RF pulse, a signal termed a free induction decay or FID can be
detected as a result of the voltage induced in the sample by the energy absorption. Eventually
the nuclear spin system relaxes to the thermal equilibrium situation necessary to fulfill the
resonance condition (Equation 1.2) are in the RF range; e.g. in a magnetic field of 14.1 T the
transition frequency no for 1H is 600 MHz, for 15N is 60.8 MHz and for 13C is 151 MHz.
As earlier stated that small bar magnet (nuclear spin) could be oriented in one of two
ways. The extent to which one orientation (energy state) is favored over the other depends on
the strength of the small nuclear magnet (proportional to gyromagnetic ratio) and the strength
of the strong magnetic field Bo in which it is placed. In practice, we do not put one nucleus in
a magnetic field. Rather a huge number (approaching Avogadro's number) of nuclei are in thesample that is placed in a magnetic field. The distribution of nuclei in the different energy
states (i.e., orientations of nuclear magnets) under conditions in which the nuclear spin
system is unperturbed by application of any RF energy is given by the Boltzmann equation
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where Nupper and Nlower represent the population (i.e., number) of nuclei in upper and
lower energy states, respectively, k is the Boltzmann constant, and T is the absolute
temperature (K). To give some idea of the consequences of increasing magnetic field on the
population of spin states, the distribution of a small number (about two million) of hydrogen
nuclei, calculated from above Equation, is shown in Figure 11. For protons in a 18.8 T
magnetic field (no = 800 MHz) at thermal equilibrium at room temperature, the population
ratio will be 0.999872. That means for every 1,000,000 nuclei in the upper energy state there
are 1,000,128 nuclei in the lower energy state. Without this small excess number of nuclei in
the lower energy state, it would not have NMR.
Figure 12- Dependence on magnetic field strength Bo of the separation of nuclear energy levels (DE) for spin I= 1/2
and the relative populations of the energy levels assuming one has approximately two million protons in the sample
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Nucleus Spin
quantum
number
Natural
Abundance
Gyromegnetic
ratio
Sensitivity Electric
quadrapol
moment
H1 1/2 99.984 26.752 100.000 -----
H2 1 0.015 4.106 0.965 0.00277
C13 1/2 1.108 6.726 1.590 -----
N15 1/2 0.365 -2.710 0.104 -----
F19 1/2 100 25.167 83.300 -----
P31 1/2 100 10.829 6.630 -----
Such a small population difference presents a significant sensitivity problem for NMR
because only the difference in populations (i.e., 128 of 2,000,128 nuclei) is detected; the
others effectively cancel one another. The low sensitivity of NMR, which has its origin here,
is probably its greatest limitation for applications to biological systems. As seen from above
Equations, the use of stronger magnetic fields will increase the population ratio and,consequently, the sensitivity. Different nuclei have different inherent sensitivities; the relative
sensitivities are listed in Table. It should be noted that other factors are also important in
detection sensitivity. For example, for macromolecules or small molecules that interact with
macromolecules, increasing magnetic field strength often increase relaxation times that can
adversely affect sensitivity (vide infra).
NMR tube diameter Minimum volume Min. Conc
H1
Min. Conc
Other Nuclei
5mm 0.25ml 0.25mM 0.5mM
8mm 1ml 0.15mM 0.3mM
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As implied by Equation the signal-to-noise (S/N) ratio in an NMR experiment will be
enhanced as the number of nuclei in the lower energy state relative to the upper energy state
increases. In addition to increasing magnetic field strength, this can be achieved by increasing
the number of nuclei in the sample, e.g., by raising the concentration (without causing
molecular aggregation) or by increasing the volume of the sample detected. For most types of
experiments, the magnetic field strength should be uniform across the sample; to the extent
that it is not, the different nuclei in a sample will achieve the Larmor condition (Equation) at
different frequencies leading to a broader signal in the NMR spectrum with a lower S/N ratio.
The geometry of the receiver coil used in detecting the NMR signal also has an effect. For
biological samples, the high dielectric constant leads to additional signal loss. Above Table
gives very approximately the amount and concentration needed for structural studies on
nucleic acids, polysaccharides and proteins in the size range 3-25 kilodaltons.
C13
NMR (7)
The spin quantum number for C12 is equal to zero. and so it does gives NMR signal.
But C13 has quantum number is and so its NMR can be observed in 23500 gauss of
magnetic field at 25.2 megacycles per second. In this technique strong pulse of radio
frequency covering a large band of frequencies which is capable to excite all resonance of
intrest at once. At the end of pulse period, the nuclei will precess freely with their
characteristic frequencies.
Each C13 resonance in organic molecule is spin coupled not only to the directly
attached proton but also to the proton which are two or four bonds away. The value of
coupling constant for C13 is over 125cps. So spectra appear as multiplets with unresolved
signals. Each signal is appears as a broad peak. The complexity in the spectrum further
increases by the overlap of multiplets due to the large number of C-H coupling.
The CNMR spectra detects1. Total number of protons2. Total number of carbon, and3. Presence of carbonyl group.
The state of hybridization is the dominating factor determining the chemical shift of a carbon
atom. Sp3 hybrid carbon atom absorbs upfield while sp2 carbon atoms absorbs at lower field
strength.
Sp2
sp sp3
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Introduction
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DEPT (7)
DEPT spectrum distinguished between CH, -CH2, -CH3 group. The novel feature in the
DEPT is variable proton pulse are set at 45, 90, 135 in three separate experiment. The
intensity of signal for individual pulses epends on the number of proton attached to theparticular carbon. In the spectra CH3 and CH shows peaks at the above side CH3 peak to the
downwards side. And quaternary carbon is nor recorded in the DEPT. but it is detected in
normal C13 NMR spectra.
D- Exchange NMR (7)
Substitution of D (Deuterium) for H (Hydrogen) results in dimunation of height of C13 signal
in a broad band decoupled spectrum. This happens because D has a spin number of a 1 and itsmagnetic moment is that of 15% H1, it will split C13 absorption into three lines. And so
because of decreased dipole dipole relaxation, Nuclear overhauser effect (NOE) is lost. There
may be chances of observing separate peak for any residual C-H. the isotope effect may also
slightly shift the absorption of the carbon atoms once removed from the deuterated carbon
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Introduction
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Impurity Profiling (11) (12) (13)
Today a majority of the drugs used are of synthetic origin. These are produced in bulk
and used for their therapeutic effects in pharmaceutical formulations. There are biologically
active chemical substances generally formulated into convenient dosage forms such as
tablets, capsules, suspensions, ointments and injectables. These formulations deliver the drug
substances in a stable, non-toxic and acceptable form, ensuring its bio-availability and
therapeutic activity. Quality, safety and efficacy of drugs Safety and efficacy of
pharmaceuticals are two fundamental issues of importance in drug therapy. The safety of a
drug is determined by its pharmacological/toxicological profile as well as the adverse effects
caused by the impurities in bulk and dosage forms. The impurities in drugs often possess
unwanted pharmacological or toxicological effects by which any benefit from their
administration may be outweighed. Therefore, it is quite obvious that the products intended
for human consumption must be characterized as completely as possible. The quality and
safety of a drug is generally assured by monitoring and controlling the impurities effectively.
Thus, the analytical activities concerning impurities in drugs are among the most important
issues in modern pharmaceutical analysis.
Origin of Impurities
Impurities in drugs are originated from various sources and phases of the synthetic
process and preparation of pharmaceutical dosage forms. A sharp difference between the
process-related impurities and degradation products is always not possible. However,
majority of the impurities are characteristic of the synthetic route of the manufacturing
process. Since there are several possibilities of synthesizing a drug, it is possible that the
same product of different sources may give rise to different impurities.
Need for Impurity Profiling
Control is more important today than ever. Until the beginning of the 20th century,
drug products were produced and sold having no imposed control. Thereupon the Food, Drug
and Cosmetic act was revised requiring advance proof of safety and various other controls for
new drugs. The impurities to be considered for new drugs are listed in regulatory documents
of the Food and Drug Administration (FDA), International Conference on the Harmonization
of the Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) andUnited States Pharmacopoeia (USP). Nevertheless, there are many drugs in existence, which
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Introduction
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have not been studied in such detail. The USP and National Formulary (NF) are the
recognized standards for potency and purity of new drugs. The most critical aspect of the
elaboration of the guidelines was the definition of the levels of impurities for identification
and qualification
Classification of Impurities (13)
Impurities can be classified in the following categories
Organic Impurities (Process and Drug Related) Inorganic Impurities Residual Solvents
Organic Impurities
It may arise during the manufacturing process and/or storage of the drug substance. They
may be identified or unidentified, volatile or nonvolatile, and include:
Starting materials By-products Intermediates Degradation products Reagents, ligands, and catalysts
Inorganic Impurities
It may derive from the manufacturing process. They are normally known and identified and
include:
Reagents, ligands, and catalysts Heavy metals
Inorganic salts Other materials (e.g., filter aids, charcoal)
Residual Solvents
Residual solvents are organic or inorganic liquids used during the manufacturing process.
Because these are generally of known toxicity, the selection of appropriate controls is easily
accomplished.
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General skim for impurity profiling
API or drug product
HPLC AnalysisConfirm peak identity
LCMS study
Preprative Isolation
Mass spectrometric study
Molecular mass and fragmentation pattern
NMR
Impurity structure and source
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Impurities decision tree for Generic drug as per USFDA (14)
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Introduction
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ICH decision tree for safety studies (14)
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Introduction
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Goals for the impurity investigation (12)
Process related impurity Degradation related impurities
Identify significant impurities Identify potential degradation product throughstress testing and stability study.
Determine origin of impurity and method forelimination or reduction
Understand degradation pathway
Establish a control system for impuritiesinvolving
1. Processing condition2. Suitable analytical methods3. specifications
Establish control system for impuritiesinvolving,
1. process condition2. analytical specification3. long term storage condition
Qualification of impurities
Qualification is the process of acquiring and evaluating data that establishes the
biological safety of an individual impurity or a given impurity profile at the levels specified.
The level of any impurity present in a drug substance that is in compliance with a USP
specification or has been adequately evaluated in comparative or in vitro genotoxicity studies
or has been evaluated via an acceptable Quanti/ative Structure Activity Relationships
(QSAR) database program is considered qualified for ANDAs. Impurities that are also
significant metabolites do not need further qualification, If data are unavailable to qualify the
proposed acceptance criteria of an impurity, studies to obtain such data may be needed when
the usual qualification threshold levels given below are exceeded..............................................
Maximum Daily dose Qualification threshold
2g/day 0.1% or 1mg/day intake
2g/day 0.05%
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Literature survey
Drug profile
The Drug substance is novel reverse transcriptase inhibitor, approved for thetreatment of HIV-1 infection alone or in combination with other anti retroviral drugs.
Parameters Description
Structure (15) N
NN
N
O
CH3
P
O
O
O
OO
O
H3CCH3O
O
O
CH3
H3C
NH2
O
HO
O
OH
Molecular formula (15) C19H30N5O10P
Molecular Weight 635.51
Solubility Freely soluble in methanol and in Dimethylformide, at 25C 2C
Category Anti-retro viral
Discription White crystalline powder
Melting point 116.86- 121.95C
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Mechanism of Action
It inhibits the activity of HIV reverse transcriptase by
competing with the natural substrate deoxyadenosine 5-
triphosphate and, after incorporation into DNA, by DNA chain
termination. Specifically, the drugs are analogues of the
naturally occurring deoxynucleotides needed to synthesize the
viral DNA and they compete with the natural deoxynucleotides
for incorporation into the growing viral DNA chain. However,
unlike the natural deoxynucleotides substrates, NRTIs and
NtRTIs (nucleoside/tide reverse transcriptase inhibitors) lack a
3'-hydroxyl group on the deoxyribose moiety. As a result,
following incorporation of an NRTI or an NtRTI, the next
incoming deoxynucleotide cannot form the next 5'-3'
phosphodiester bond needed to extend the DNA chain. Thus,
when an NRTI or NtRTI is incorporated, viral DNA synthesis is
halted, a process known as chain termination. All NRTIs and
NtRTIs are classified as competitive substrate inhibitors.
Adverse effects
Sever/ fatal lever problem Lactic acidosis Nausea Vomiting Pale stools Dark urine Yellowing eyes/skin Unusual tiredness Drowsiness
Precautions Contraindicated in Hepatitis B
Uses Treatment of HIV infection (AIDS)
Table 4- Drug Profile
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Literature survey
1. US Patent Publication no. US 2009/0270352 A1, Publication date, Oct 2009: Thepresent invention shows the different claims for the invented drug substance related
with its crystalline form type, DSC pattern and other important parameters for ANDAfilling.
2. Authorized USP Pending Monograph, Version 1 for the drug substance has shownthe RPHPLC method for API.
3. Sonal Desai, Archita Patel, SY Gabhe: has developed A simple isocratic reversedphase high performance liquid chromatography was used to separate three impuritiespresent in the sample of 8-chlorotheophylline. LC-MS was used for the
characterization of impurities. Based on mass spectral data, the structures of these
impurities were characterized as 3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione
(impurity I), 3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione (impurity II) and
isomer of 8-chloro-1,3-dimethyl-2,6(3H,1H)-purinedione (impurity III).
4. Dunge Ashenafi, Varalaxmi Chintam et al: The study describes the developmentand validation of a selective liquid chromatographic (LC) method for the analysis of
tenofovir disoproxil fumarate (TDF) and its related substances. The gradient method
uses a base deactivated C18 column (Hypersil BDS column; 25 cm4.6mm I.D.)
maintained at a temperature of 301C. The mobile phases consist of acetonitrile,
tetrabutylammonium/phosphate buffer pH 6.0 and water (A; 2:20:78 v/v/v) and (B;
65:20:15 v/v/v). The flow rate is 1.0 mL/min and UV detection is performed at 260
nm. The method is proved to be robust, precise, sensitive and linear between 0.1
mg/mL and 0.15 mg/mL. The limit of detection and limit of quantification are 0.03
and 0.1 mg/ mL, respectively. The method was successfully applied to the
quantification of related substances and assay of commercial TDF samples.
5. Pei Xi Zhu et al: has done a study on Characterization of impurities in the bulk druglisinopril by liquid chromatography/ion trap spectrometry and Two trace impurities in
the bulk drug lisinopril were detected by means of high-performance liquid
chromatography coupled with mass spectrometry (HPLC/MS) with a simple andsensitive method suitable for HPLC/MSn analysis. The fragmentation behavior of
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lisinopril and the impurities was investigated, and two unknown impurities were
elucidated as named 2-(6-amino-1-(1-carboxyethylamino)-1-oxohexan-2-ylamino)-4
phenylbutanoic acid and 6-amino-2-(1-carboxy-3-phenylpropylamino)-hexanoic acid
on the basis of the multi-stage mass spectrometry and exact mass evidence. The
proposed structures of the two unknown impurities were further confirmed by nuclear
magnetic resonance (NMR) experiments after preparative isolation.
6. Reguri buchi redy et al: has done a work on Identification and Characterization ofPotential Impurities in Raloxifene Hydrochloride and During the synthesis of the bulk
drug Raloxifene hydrochloride, eight impurities were observed, four of which were
found to be new. All of the impurities were detected using the gradient high
performance liquid chromatographic (HPLC) method, whose area percentages ranged
from 0.05 to 0.1%. LCMS was performed to identify the mass number of these
impurities, and a systematic study was carried out to characterize them. These
impurities were synthesized and characterized by spectral data, subjected to co-
injection in HPLC, and were found to be matching with the impurities present in the
sample. Based on their spectral data (IR, NMR, and Mass.
7. Gosula Venkat ram reddy et al: has done a research work on separation,identification and structural elucidation of new impurity in the drug substance of
Amlodipine Maleate using LCMS/MS, NMR and IR and they have found Amlodipine
maleate is a maleate salt of 3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-
chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate. An unknown
impurity at m/z 392.2 for [M+H]+ ion has been detected during the accelerated
stability analysis (40 C /75 % RH) of amlodipine maleate drug substance by reverse-
phase high performance liquid chromatographymass spectrometry (RP-HPLC-MS).MS and MS/MS spectra of amlodipine maleate and unknown impurity are obtained
using HPLC-MS/MS equipped with positive electrospray ionization (ESI). The
nuclear magnetic resonance (NMR) and infrared (IR) spectra of the unknown
impurity are recorded after isolation of the impurity by preparative HPLC. Based on
MS, NMR and IR spectral data, the structure of the unknown impurity was proposed.
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8. Dennis J Milanowaski and Ulla Mocek: has explained the trace impurityidentification with a combination of spectroscopic and spectrometric techniques and
explained the outline generally followed for the isolation and structural elucidation of
any impurities in the drug substances by using LCMS and preparative HPLC and
NMR.
9. Sandor gorog: A review article on The role of impurity profiling in drug research ,development and producton. Has explained the sources of impurities and methods for
estimating and identification of impurities.
10.Guidance for Industry ANDAs: Impurities in Drug substances by US Department ofhealth and human services Food and drug Administration This guidance providesrecommendations for including information in abbreviated new drug applications
(ANDAs) and supporting drug master files (DMFs) on the identification and
qualification of impurities in drug substances produced by chemical syntheses for
both monograph and non-monograph drug substances. nnnnnnnnnnnnnnnnnnnnnnnnn
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Research Envisaged
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Research Envisaged
The presence of impurities or in a drug substance can have a significant impact on the
quality and safety of the drug product. Impurities in drug substance can arise from
degradation of API itself, which is related to stability of pure API during storage, and the
manufacturing process including the chemical synthesis. Process impurities, includes
unreacted starting materials, chemical derivatives of impurities, synthetic by-products and
degradation products etc. In addition to stability, which is a factor in shelf-life of API, purity
of API is also necessary for commercially. Purity standards are established to ensure that API
is free of impurities as possible and thus safe for clinical use. It is, therefore, essential to
isolate and characterize unidentified impurities present in the drug sample.
Objective of Work
Literature survey of drug substance (Pharmacopoeia, Research articles), andchromatographic separation, characterization tools, impurity isolations.
Details of instrumentations techniques employed for chromatographic separations andstructural characterizations.
API characterization using spectroscopic techniques.
Method development (Chromatographic parameters) Impurity isolation and characterization.
Plan of work
Literature survey Spectral characterization of API Review of developed HPLC method Develop simple and precise method for impurity identification on LCMS Isolation of impurities using preparative HPLC Identification and structural elucidation and probable sources of impurities present in
API
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Experimental Work
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Experimental Work
List of Instruments
Instrument Make Model
UV Spectrophotometer Shimadzu UV-1700
FTIR Shimadzu FTIR-8400
Balance Sartorius CPA2250 (M)
LCMS Thermo Agilent Max 200 Series
XRD PANalytical Xpert Pro
Preparative HPLC Shimadzu LC-8A
NMR Bruker AVANCE-II
DSC Mettler Toledo DSC 822e
pH Meter Lab India PICO Plus
Milli-Q-H2O System Elix NA
Sonicator PCI 14.7 L-300/CC/DTC
Melting Point Appratus Scientific ----
Table 5- List of Instruments
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List of Chemicals
Chemical Name Manufacturer Grade
Acetoitrile Rankem HPLC
Methanol Rankem HPLC
Water ----- Milli-Q
DMSO-d6
Hydrogen Peroxide Thomas Baker AR
Butanol Rankem HPLC
Trifluoroacetic Acid Rankem AR
Table 6 List of Chemicals
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Experimental Work
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Identification and Characterization of API
Ultra-Violet Spectroscopy
Preparation of Stock Solution (1000 ppm) - Weigh accurately about 100 mg of API andtransferred it to 100 ml volumetric flask. Add 25 ml of diluent and sonicate it to dissolve.
Make up the volume with diluent.
Dilution 1 (100 ppm) - Pipette out 1 ml of standard stock solution and transfer to 10 ml
volumetric flask. Make up the volume with diluent.
Final Solution (6 ppm) - Pipette out 0.6 ml of above sample (Dilution 1) and transfer it to 10
ml volumetric flask. Make up the volume with diluent.
Infrared Spectroscopy
Background scanning- Triturate about 10 mg of dry, finely powdered potassium bromide
(IR) in mortar- pestle and spread it uniformly in a sample holder and compress it with some
pressure and record the spectra in IR Range.
Sample Preparation- Triturate about 1 mg of API with approximately 300 mg of dry, finely
powdered potassium bromide (IR). Grind the mixture thoroughly, dry, finely powderedpotassium bromide IR.
X-Ray Diffraction
Sample Preparation- The sample was loaded by back-loading method.
Scanning- Sample is scanned for 2- 50 angle, with the speed of 50 second per step with
the step size of 0.0170 angle
Scan Axis- Gonio
Scan Type- Continuous
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Differential Scanning Calorimeter
Sample Preparation- Weigh 3-5 mg of API and transfer it to 40 l Aluminium crucible.
Make two holes to the lid to escape volatile gas that evolves on thermal decomposition.
Crimp the lid with crucible using crimper.
Scanning- Sample is analyzed as per below mentioned parameters
Scanning range- 35- 250 C
Heating rate- 10C/ min
Nitrogen Flow- 60cc/min
Mass Spectroscopy
Mode- ESI
Nuclear Magnetic Resonance
Sample Preparation- Prepare sample using DMSO-d6 as solvent and studied with H1 NMR,
C13
NMR, D-Exchange, DEPT, and COSY (Correlation Spectroscopy).
Development of Liquid Chromatography Method Suitable for LCMS
Sample Preparation- Accurately weigh sample. Transfer to 25 ml volumetric flask. Addabout 15 ml of diluents, sonicate to dissolve and make up the volume with diluents.
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Experimental Work
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HPLC Method
Mobile Phase Mobile phase A: Buffer (disodium hydrogen phosphate)
Mobile phase B: Methanol: Butanol
Diluents Mobile phase
Column ODS 5m (250mm 4.6mm)
Flow rate 0.7 ml/min
Detector UV max = 260nm
Sample injection 20l
Pump Gradient
Gradient Programme
Time %A %B
0 70 27.5: 2.5
08 70 27.5: 2.5
15 65 32.5: 2.5
40 65 32.5: 2.5
55 52 45.5: 2.5
60 52 45.5: 2.5
75 40 57.5: 2.5
80 40 57.5: 2.5
Table 7- HPLC Method
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Experimental Work
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Method for LCMS
Trial 1
Mobile Phase Buffer- 0.01M Ammonium Acetate pH= 6Mobile Phase A- Buffer
Mobile Phase B- Methanol
Diluents Methanol
Column ODS 5m (250mm 4.6mm)
Flow rate 1.5ml/min
Detector UV max = 260nm
Sample injection 20l
Pump Gradient
Gradient Programme
Time %A %B
0 100 00
10 100 00
19 80 20
43 80 20
51 40 40
57 40 40
63 0 100
80 0 100
Table 8- Method for LCMS (Trial 1)
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Experimental Work
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Trial 2
Mobile Phase Buffer- 0.01M Ammonium Acetate pH= 6
Mobile Phase A- Buffer
Mobile Phase B- Methanol : Butanol
Diluents Mobile phase
Column ODS 5m (250mm 4.6mm)
Flow rate 0.7 ml/min
Detector UV max = 260nm
Sample injection 20 l
Pump Gradient
Gradient Programme
Time %A %B
0 60 37.5: 2.508 60 37.5: 2.5
15 55 42.5: 2.5
40 55 42.5: 2.5
55 40 57.5: 2.5
60 40 57.5: 2.5
75 35 62.5: 2.5
80 35 62.5: 2.5
Table 9- Method for LCMS (Trial 2)
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Experimental Work
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Trial 3
Mobile Phase Buffer- 0.01M Ammonium Acetate pH= 6
Mobile Phase A- Buffer
Mobile Phase B- Methanol : Butanol
Diluents Buffer, Methanol, Butanol
Column ODS 5m (250mm 4.6mm)
Flow rate 0.7ml/min
Detector UV max = 260nm
Sample injection 20l
Pump Gradient
Gradient Programme
Time %A %B
0 70 27.5: 2.5
8 70 27.5: 2.5
15 64 33.5: 2.5
40 64 33.5: 2.5
45 52 45.5: 2.5
55 52 45.5: 2.5
60 40 57.5: 2.5
75 40 57.5: 2.5
80 40 57.5: 2.5
Table 10- Method for LCMS (Trial 3)
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Final Method
Mobile Phase Buffer- 0.01M Ammonium Acetate PH= 6
Mobile Phase A- Buffer
Mobile Phase B- Methanol : Butanol ()
Diluents Buffer, Methanol, Butanol
Column ODS 5m (250mm 4.6mm)
Flow rate 0.7ml/min
Detector UV max = 260nm
Sample injection 20l
Pump Gradient
Gradient Programme
Time %A %B
0 70 27.5: 2.5
8 70 27.5: 2.5
15 64 33.5: 2.5
40 64 33.5: 2.5
45 52 45.5: 2.5
55 52 45.5: 2.5
60 40 57.5: 2.5
75 40 57.5: 2.5
Table 11- Method for LCMS (Final Method)
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Experimental Work
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Mass Identification of required Peaks
We are required to identify the masses of impurities which show peaks at 28.27mins
and 59.34mins in LC Chromatogram and masses were identified.
Preparative Isolation of Impurities
NMR of impurities
Impurities structure were identified by H1NMR and correlated it with obtained Mass.
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Result and Discussion
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Result and Discussion
A key component of the overall quality of drug is control of impurities, as it may affect drug
safety and efficacy. The identification of impurities presents a significant challenge to the
analyst. Analytical science is developing rapidly and provides increasing opportunity toidentify structures and so origin of these impurities. The present study deals with the study of
these impurities and isolating and confirming it. It involves the following steps.
Characterization of API Development of HPLC Method Identification of Impurities Isolation of Impurities by Preparative HPLC Identification of structures of isolated impurities
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Result and Discussion
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Characterization of API
UV absorbance
UV Spectra Observation
The absorbance maxima (max) is observed at the 260nm.
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Result and Discussion
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XRD (X-ray Diffraction)
Figure 13 Powder X-Ray diffraction Pattern of API
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Result and Discussion
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X-ray Difractogram Observation
The major peaks are present at the 2 position of 4.9, 10.2, 7.7, 8.0 0.2 are present inthe difractogram of API
Pos. [2 Th] Height [cts] d- spacing Area Rel. int [%]
5.0213 5996.61 17.59937 494.76 100
5.4576 436.60 16.19321 50.43 7.28
6.9601 166.20 12.70061 27.43 2.77
7.8207 151.87 11.30486 15.04 2.53
8.1107 325.52 10.90124 32.23 5.43
8.9297 135.59 9.90314 22.37 2.26
10.3157 1008.09 8.57547 116.44 16.81
10.6131 810.60 8.33588 80.26 13.52
10.8877 459.23 8.12623 53.04 7.66
11.4597 464.86 7.72186 53.70 7.75
11.9843 181.30 7.38499 17.95 3.02
12.7736 496.86 6.93041 40.94 8.27
13.4074 526.61 6.60416 121.66 8.78
14.3329 437.28 6.17972 57.73 7.29
15.0015 1298.35 5.90580 149.97 21.65
15.4553 373.61 5.73338 55.49 6.23
15.8061 434.68 5.60694 57.38 7.25
16.2035 451.05 5.47030 52.10 7.52
16.8069 369.89 5.27524 30.52 6.17
17.3574 162.13 5.10917 21.40 2.70
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Result and Discussion
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Pos. [2 Th] Height [cts] d- spacing Area Rel. int [%]
18.3761 1084.26 4.82816 322.05 18.08
18.7588 343.65 4.73052 45.37 5.73
19.1921 334.05 4.62468 44.10 5.57
20.0450 3383.71 4.42979 837.53 56.43
21.2241 676.02 4.18628 89.24 11.27
22.0415 1773.46 4.03286 321.91 29.57
22.7868 873.02 3.90260 72.03 14.56
23.1145 554.62 3.84800 91.52 9.25
24,0563 590.46 3.69946 136.41 9.85
24.2917 755.79 3.66413 99.71 12.60
25.0838 4688.11 3.55020 773.60 78.18
25.5401 901.05 3.48779 178.42 15.03
26.2522 118.94 3.39478 31.40 1.98
27.2084 236.55 3.27761 46.84 3.94
27.9591 284.45 3.19129 42.24 4.74
30.1789 1180.55 2.96142 233.77 19.69
31.5818 73.19 2.83300 19.32 1.22
32.7119 115.30 2.73766 22.83 1.92
35.3803 224.06 2.53707 51.76 3.74
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Result and Discussion
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Differential Scanning Calorimeter (DSC)
Figure 14- Themogram of API
DSC Observation
With the DSC Scanning of API, it is observed that the chemical is endothermic in
nature and it shows endothermic peak at 118.89 C.
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Result and Discussion
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Infrared Spectroscopy (IR)
Figure 15- IR Spectra of API
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Result and Discussion
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IR Observations
Sr. No. Wavenumber cm- Assignments
1 3198.08 -OH, -NH Broad
2 2985.91 -CH Aliphatic
3 1759.14 C=O
4 1685.84 C=O
5 1273.06 P=O
6 1103.32 C-O
7 1033.88 C-O
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Result and Discussion
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Nuclear Magnetic Resonance (NMR)
H1NMR
Figure 16- H1NMR of API
H1NMR Observations
N
NN
N
O
CH3
P
O
O
O
OO
O
H3CCH3O
O
O
CH3
H3C
NH2
O
HO
O
OH
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Result and Discussion
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Table 12- Observations of H
1
NMR of API
Sr. No Chemical Shift Proton No. of Protons Multiplicity
1 1.057-1.078 A 3 Doublet
2 1.224-1.245 B 12 Doublet
3 3.920-4.054 C 3 Multiplets
4 4.140-4.305 D 2 Multiplets
5 4.755-4.885 E 2 Multiplets
6 5.487-5.601 F 4 Multiplets
7 6.640 G 2 Singlet
8 7.273 I 2 Broad Singlet
9 8.044 J 1 Singlet
10 8.152 K 1 Singlet
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Result and Discussion
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Result and Discussion
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D- Exchange NMR
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Result and Discussion
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C13
NMR
Figure 17 C13NMR of API
C13
NMR Observations
N
NN
N
O
CH3
P
O
O
O
OO
O
H3C CH3O
O
O
CH3
H3C
NH2
O
HO
O
OH
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Result and Discussion
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Sr. No Chemical Shift No. of Carbons Assignments
1 16.890 1 Aliphatic -CH3
2 21.502 4 Aliphatic -CH3
3 46.802 1 AliphaticCH2
4 61.218, 63.404 1 AliphaticCH2
5 73.094 2 AliphaticCH
6 76.056, 76.217 1 AliphaticCH
7 84.368, 84,449 2 AliphaticCH2
8 118.540 1 Quaternary -C
9 134.228 2 OlefenicCH
10 141.550 1 TertiaryCH
11 149.999 1 QuaternaryC
12 152.538 1 TertiaryCH
13 152.791, 152.803 2 QuaternaryC
14 156.079 1 QuaternaryC
15 166.259 2 QuaternaryCO
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Result and Discussion
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Distorytionless Enhancement by Polarization Transfer (DEPT)
Observations from DEPT NMR
In the API, four secondary hydrogen are present.
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Result and Discussion
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Correlation Spectroscopy (COSY)/ 2D NMR
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Result and Discussion
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Mass Spectrometry (MASS)
Mass Spectrometry Observations
Molecular Mass
(Theoretical Data)
Molecular Mass
(Experimental Data)
Interpretation
519.4
(Excluding Fumaric acid Moiety)
520 M+H
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Result and Discussion
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HPLC Chromatogram
And from the above chromatogram we are required to identify the impurities eluting at the28.124 mins and 60.020mins of retention time.
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Result and Discussion
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LCMS Method development
Trial 2
Trial 3
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Result and Discussion
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Final Method
Mass identification through X-Caliber
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Result and Discussion
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Preparative isolation of impurities
Impurity 1
Trial 1
Trial 2
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Result and Discussion
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Purity on Analytical HPLC
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Result and Discussion
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Mass identification through DI (Direct Injection)
Observations from Mass Spectra
Molecular Mass
(Experimental Data)
Interpretation
817 (M-H)
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Result and Discussion
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H1NMR of impurity 1
Observations from NMR Spectra
NN
N
O
CH3
P
HO O
O
O
O
O
H3C
CH3
NH
NH
NN
N
N
O
POH
O
O
O
O
O
CH3
CH3
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Result and Discussion
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Sr. No Chemical Shift Protons Number of
Protons
Multiplicity
1 0.923- 0.943 A 6 Doublet
2 1.155- 1.176 B 12 Doublet
3 3.283- 3.326 C 4 Multiplet
4 3.794- 3.887 D 2 Multiplet
5 4.125- 4.295 E 4 Multiplet
6 4.652- 4.777 F 2 Septet
7 5.296- 5.386 G 6 Multiplet
8 8.017 I 2 Broad Peak
9 8.290 J 4 Broad Peak
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Result and Discussion
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Result and Discussion
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Impurity 2
Preparative isolation
Purity on HPLC
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Result and Discussion
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Mass identification through Direct injection (DI)
Observations from Mass spectra
Molecular Mass Interpretation
933 (M-H)
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Result and Discussion
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H1NMR of Impurity 2
Observations from H1NMR Spectra
N
NN
N
O
CH3
P
O O
O
O
O
O
H3C
CH3
O
O
O
CH3
H3C
NH
NN
N
N
O
H3C
POH
O
O
O
O
O
CH3
CH3
NH
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Result and Discussion
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Sr. No Chemical Shift Number of
Protons
Multiplicity
1 0.908- 0.927 3 Doublet
2 1.042- 1.062 3 Doublet
3 1.157- 1.178 6 Doublet
4 1.200- 1.220 12 Doublet
5 3.360- 3.369 2 Multiplet
6 3.815- 3.866 1 Multiplet
7 3.934- 3.981 3 Multiplet
8 4.120- 4.304 4 Multiplet
9 4.653- 4.757 1 Multiplet
10 4.772- 4.862 2 Multiplet
11 5.298- 5.385 2 Multiplet
12 5.402 2 Broadpeak
13 5.471- 5.561 4 Multiplet
14 8.040- 8.295 6 Broadpeak
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Result and Discussion
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Summary and Conclusion
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SUMMARY AND CONCLUSION
Characterization and Impurity profile is the description of identified and unidentifiedimpurities present in new drug substances. The entire study consisted of the following steps:
1. Identification and Characterization of API
2. Development of LCMS Method
3. Degradation Studies
4. Isolation of Impurities Using Preparative HPLC
5. Identification and Characterization of Isolated Impurities
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Bibliography
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Bibliography
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