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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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    Introduction

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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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    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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    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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    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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    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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    ICH decision tree for safety studies (14)

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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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    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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    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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    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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    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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    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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    Experimental Work

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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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