Thesis Draft FinalM

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management
Introduction 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 safety and 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; Classical methods and Instrumental methods Classical Methods (1) 1. Volumetric method: It is based on the determination of a solution of known strength required to complete a chemical reaction with the substance under analysis 2. Gravimetric method: In this method of analysis, the assay results generally obtained either 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 new products. 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 Spectrophotometry Nuclear 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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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). Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management 3|Page
Introduction
Figure 1- Retention Process in Chromatography
High Performance Liquid Chromatography (2) (4) (5) In HPLC, for separation of individual components, the sample is being introduced into 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 operation of 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 in the 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
Retention by interaction of the Retention by interaction of the stationary phases polar surface stationary
Mechanism
phases
non-polar
with polar parts of the sample hydrocarbon chain with non-polar molecules. parts of sample
bonded
Stationary Phase
siloxane
with
polar bonded siloxane with non-polar
functional group like SiO2, Al2O3, - functional groups like n- octadecyl NH2, -CN, -NO2, - Diol (C-18) or n- octyl (C-8), ethyl, phenyl, -(CH2) n-diol, (CH2) n-CN molecules.
Nonpolar solvents like heptane, Polar

Mobile Phase
solvents
like
methanol, or buffer
hexane, cyclohexane, chloroform, acetonitrile, ethyl ether, dioxane
water
(Sometimes with additives of THF or dioxane).
Elution Order
Least polar components are eluted first
Most polar components are eluted first
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 2000 2000- 4000 Region Near UV region 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 matter can 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 graph obtained from the DSC is called the Thermogram. Thermogram usually shows various Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management 10 | P a g e
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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 the elucidation 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) Near IR region (vibration region) Far IR region (rotation region)
1.2 to 2.5
2.5 to 25
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 vibrations 2. 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. Symmetric 2. Asymmetric
Symmetric
Asymmetric
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Bending vibrations: 1. 2. 3. 4. Scissoring Rocking Twisting Wagging
Scissoring
Wagging
Rocking 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 sources 2. Monochromators 3. Sample cells 4. Detectors
Twisting
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IR radiation sources (2)
Glober sources
Incandesc ent lamp
IR radiation sources
Mercury arc
Nnernst glower
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 Monochromators 2. 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.
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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 is fixed 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
Golay cells
Photoconductivity cells
IR Detectors
Bolometers
Thermal detectors
Figure 7- 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 C-H
Mode Stretch Stretch (2v) Stretch (3v) Stretch (C) Bend in plane Bend out of plane Rocking
Wavenumber (cm-1) 2700 - 3300 5600 6300 8300 9000 4200 5000 1300 1500 800 830 600 900 800 - 1200 900 - 1300 800 1200 1600 1700
C-C C-O C-C
Stretch Stretch Stretch
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Bond C=O Mode Stretch Stretch (2v) Stretch (3v) C=N C=C C=N C-F C-Cl C-Br C-I O-H Stretch Stretch Stretch Stretch Stretch Stretch Stretch Stretch Stretch (2v) N-H Stretch Stretch (2v) Stretch (3v) Stretch (C) Bending rocking Wavenumber (cm-1) 1600 1900 3300 3600 5000 5300 1600 1700 2100 2400 2100 2400 1000 1400 600 800 500 600 500 3000 3700 6700 7100 3000 3700 6300- 7100 9000 10000 4800 5300 1500 1700 700 900
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. Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management 19 | P a g e
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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. This tiny 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 including 1H,
13 15 19 31
C,
N,
F, and
P; so all those which have nuclear spin quantum
number 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, zero.
14 16
N and
O) do not have a nuclear spin because of their spin quantum no is
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 the signal displayed as a free 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 lists the 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 the sample 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 H2 C13 N15 F19 P31
1/2 1 1/2 1/2 1/2 1/2
99.984 0.015 1.108 0.365 100 100
26.752 4.106 6.726 -2.710 25.167 10.829
100.000 0.965 1.590 0.104 83.300 6.630
----0.00277 -----------------
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 H
1
Min. Conc Other Nuclei 0.5mM 0.3mM
5mm 8mm
0.25ml 1ml
0.25mM 0.15mM
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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 detects 1. Total number of protons 2. Total number of carbon, and 3. 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 the particular 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 C 13 signal in a broad band decoupled spectrum. This happens because D has a spin number of a 1 and its magnetic 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) and United States Pharmacopoeia (USP). Nevertheless, there are many drugs in existence, which Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management 28 | P a g e
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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 Analysis Confirm 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 Identify significant impurities Degradation related impurities Identify potential degradation product through stress testing and stability study.
Determine origin of impurity and method for Understand degradation pathway elimination or reduction Establish a control system for impurities Establish control involving involving, 1. Processing condition 2. Suitable analytical methods 3. specifications system for impurities
1. process condition 2. analytical specification 3. 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 2g/day 2g/day
Qualification threshold 0.1% or 1mg/day intake 0.05%
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Literature survey
Literature survey
Drug profile The Drug substance is novel reverse transcriptase inhibitor, approved for the treatment of HIV-1 infection alone or in combination with other anti retroviral drugs.
Parameters
Description
H3C CH3 O O
NH2 N
H3C
CH3
Structure (15)
OH O O P O O HO O O
CH3
Molecular formula (15)

Molecular Weight
C19H30N5O10P
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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Literature survey
It inhibits the activity of HIV reverse transcriptase by competing with the natural substrate deoxyadenosine 5triphosphate 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
Mechanism of Action
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
Literature survey 1. US Patent Publication no. US 2009/0270352 A1, Publication date, Oct 2009: The present invention shows the different claims for the invented drug substance related with its crystalline form type, DSC pattern and other important parameters for ANDA filling.
2. Authorized USP Pending Monograph, Version 1 for the drug substance has shown the RPHPLC method for API.
3. Sonal Desai, Archita Patel, SY Gabhe: has developed A simple isocratic reversed phase high performance liquid chromatography was used to separate three impurities present 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 development and 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 drug lisinopril 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 and sensitive method suitable for HPLC/MSn analysis. The fragmentation behavior of Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management 36 | P a g e
Literature survey
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 of Potential 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 coinjection 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-(2chlorophenyl)-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 reversephase 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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Literature survey
8. Dennis J Milanowaski and Ulla Mocek: has explained the trace impurity identification 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 of health and human services Food and drug Administration This guidance provides recommendations 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
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), and chromatographic separation, characterization tools, impurity isolations. Details of instrumentations techniques employed for chromatographic separations and structural 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
Experimental Work
List of Instruments Instrument Make Model
UV Spectrophotometer FTIR
Shimadzu Shimadzu
UV-1700 FTIR-8400
Balance LCMS
Sartorius Thermo Agilent
CPA2250 (M) Max 200 Series Xpert Pro LC-8A
XRD Preparative HPLC
PANalytical Shimadzu
NMR DSC
Bruker Mettler Toledo
AVANCE-II DSC 822e
pH Meter Milli-Q-H2O System
Lab India Elix
PICO Plus NA
Sonicator
PCI
14.7 L-300/CC/DTC
Melting Point Appratus
Scientific
----
Table 5- List of Instruments
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Experimental Work
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
Identification and Characterization of API
Ultra-Violet Spectroscopy Preparation of Stock Solution (1000 ppm) - Weigh accurately about 100 mg of API and transferred 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 powdered potassium 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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Experimental Work
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. Add about 15 ml of diluents, sonicate to dissolve and make up the volume with diluents.
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Experimental Work
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
Time
%A
%B
0 08 Gradient Programme 15 40 55 60 75 80
Table 7- HPLC Method
70 70 65 65 52 52 40 40
27.5: 2.5 27.5: 2.5 32.5: 2.5 32.5: 2.5 45.5: 2.5 45.5: 2.5 57.5: 2.5 57.5: 2.5
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Experimental Work
Method for LCMS Trial 1 Mobile Phase Buffer- 0.01M Ammonium Acetate pH= 6 Mobile 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
Time
%A
%B
0 10 Gradient Programme 19 43 51 57 63 80
100 100 80 80 40 40 0 0
00 00 20 20 40 40 100 100
Table 8- Method for LCMS (Trial 1)
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Experimental Work
Trial 2 Mobile Phase Buffer- 0.01M Ammonium Acetate pH= 6 Mobile Phase A- Buffer Mobile Phase B- Methanol : Butanol
Diluents Mobile phase
Column Flow rate Detector Sample injection Pump Time

0 08 15
ODS 5m (250mm 4.6mm) 0.7 ml/min UV max = 260nm 20 l Gradient %A

60 60 55 55 40 40 35 35
%B
37.5: 2.5 37.5: 2.5 42.5: 2.5 42.5: 2.5 57.5: 2.5 57.5: 2.5 62.5: 2.5 62.5: 2.5
Gradient Programme
40 55 60 75 80
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Experimental Work
Trial 3 Mobile Phase Buffer- 0.01M Ammonium Acetate pH= 6 Mobile Phase A- Buffer Mobile Phase B- Methanol : Butanol
Diluents
Buffer, Methanol, Butanol ODS 5m (250mm 4.6mm) 0.7ml/min UV max = 260nm 20l Gradient Time 0 8 15 %A 70 70 64 64 52 52 40 40
40
Column Flow rate Detector Sample injection Pump
%B 27.5: 2.5 27.5: 2.5 33.5: 2.5 33.5: 2.5 45.5: 2.5 45.5: 2.5 57.5: 2.5 57.5: 2.5 57.5: 2.5
Gradient Programme 40 45 55 60 75
80
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Experimental Work
Final Method Mobile Phase Buffer- 0.01M Ammonium Acetate PH= 6 Mobile Phase A- Buffer Mobile Phase B- Methanol : Butanol ()
Diluents
Buffer, Methanol, Butanol ODS 5m (250mm 4.6mm) 0.7ml/min UV max = 260nm 20l Gradient Time 0 %A 70 70 64 64 52 52 40 40 %B 27.5: 2.5 27.5: 2.5 33.5: 2.5 33.5: 2.5 45.5: 2.5 45.5: 2.5 57.5: 2.5 57.5: 2.5
Column Flow rate Detector Sample injection Pump
Gradient Programme
8 15 40 45 55 60 75
Table 11- Method for LCMS (Final Method)
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Experimental Work
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

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 to identify 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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Characterization of API
UV absorbance
UV Spectra Observation The absorbance maxima (max) is observed at the 260nm.
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XRD (X-ray Diffraction)
Figure 13 Powder X-Ray diffraction Pattern of API
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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 in the difractogram of API
Pos. [2 Th] 5.0213 5.4576 6.9601 7.8207 8.1107 8.9297 10.3157 10.6131 10.8877 11.4597 11.9843 12.7736 13.4074 14.3329 15.0015 15.4553 15.8061 16.2035 16.8069 17.3574 Height [cts] 5996.61 436.60 166.20 151.87 325.52 135.59 1008.09 810.60 459.23 464.86 181.30 496.86 526.61 437.28 1298.35 373.61 434.68 451.05 369.89 162.13 d- spacing 17.59937 16.19321 12.70061 11.30486 10.90124 9.90314 8.57547 8.33588 8.12623 7.72186 7.38499 6.93041 6.60416 6.17972 5.90580 5.73338 5.60694 5.47030 5.27524 5.10917 Area 494.76 50.43 27.43 15.04 32.23 22.37 116.44 80.26 53.04 53.70 17.95 40.94 121.66 57.73 149.97 55.49 57.38 52.10 30.52 21.40 Rel. int [%] 100 7.28 2.77 2.53 5.43 2.26 16.81 13.52 7.66 7.75 3.02 8.27 8.78 7.29 21.65 6.23 7.25 7.52 6.17 2.70
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Pos. [2 Th] 18.3761 18.7588 19.1921 20.0450 21.2241 22.0415 22.7868 23.1145 24,0563 24.2917 25.0838 25.5401 26.2522 27.2084 27.9591 30.1789 31.5818 32.7119 35.3803 Height [cts] 1084.26 343.65 334.05 3383.71 676.02 1773.46 873.02 554.62 590.46 755.79 4688.11 901.05 118.94 236.55 284.45 1180.55 73.19 115.30 224.06 d- spacing 4.82816 4.73052 4.62468 4.42979 4.18628 4.03286 3.90260 3.84800 3.69946 3.66413 3.55020 3.48779 3.39478 3.27761 3.19129 2.96142 2.83300 2.73766 2.53707 Area 322.05 45.37 44.10 837.53 89.24 321.91 72.03 91.52 136.41 99.71 773.60 178.42 31.40 46.84 42.24 233.77 19.32 22.83 51.76 Rel. int [%] 18.08 5.73 5.57 56.43 11.27 29.57 14.56 9.25 9.85 12.60 78.18 15.03 1.98 3.94 4.74 19.69 1.22 1.92 3.74
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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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Infrared Spectroscopy (IR)
Figure 15- IR Spectra of API
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IR Observations
Sr. No. 1 2 3 4 5 6 7
Wavenumber cm-1 3198.08 2985.91 1759.14 1685.84 1273.06 1103.32 1033.88
Assignments -OH, -NH Broad -CH Aliphatic C=O C=O P=O C-O C-O
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Nuclear Magnetic Resonance (NMR) H1NMR
Figure 16- H1NMR of API
H1NMR Observations
H3C CH3 O O
NH2 N
H3C
CH3
OH O O P O O HO O O
CH3
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Sr. No
Chemical Shift
Proton
No. of Protons
Multiplicity
1.057-1.078
Doublet
1.224-1.245
12
Doublet
3.920-4.054
Multiplets
4.140-4.305
Multiplets
4.755-4.885
Multiplets
5.487-5.601
Multiplets
6.640
Singlet
7.273
Broad Singlet
8.044
Singlet
10
8.152
Singlet
Table 12- Observations of H1NMR of API
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D- Exchange NMR
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C13 NMR
Figure 17 C13NMR of API
C13NMR Observations
H3C CH3 O O
NH2 N
H3C
CH3
OH O O P O O HO O O
CH3
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Sr. No
Chemical Shift
No. of Carbons
Assignments
16.890
Aliphatic -CH3
21.502
Aliphatic -CH3 Aliphatic CH2 Aliphatic CH2 Aliphatic CH
46.802
61.218, 63.404
73.094
76.056, 76.217
Aliphatic CH Aliphatic CH2
84.368, 84,449
118.540
Quaternary -C Olefenic CH
134.228
10
141.550
Tertiary CH
11
149.999
Quaternary C
12
152.538
Tertiary CH
13
152.791, 152.803
Quaternary C
14
156.079
Quaternary C
15
166.259
Quaternary CO
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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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Correlation Spectroscopy (COSY)/ 2D NMR
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Mass Spectrometry (MASS)
Mass Spectrometry Observations
Molecular Mass (Theoretical Data)
Molecular Mass (Experimental Data)
Interpretation
519.4 520 (Excluding Fumaric acid Moiety) M+H
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HPLC Chromatogram
And from the above chromatogram we are required to identify the impurities eluting at the 28.124 mins and 60.020mins of retention time.
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LCMS Method development Trial 2
Trial 3
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Final Method
Mass identification through X-Caliber
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Preparative isolation of impurities Impurity 1 Trial 1
Trial 2
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Purity on Analytical HPLC
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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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H1NMR of impurity 1
Observations from NMR Spectra

N N
N N
NH CH3
P O
OH
NH N
H3 C O O HO O P O O O
O O CH3 O CH3
CH3
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Sr. No
Chemical Shift
Protons
Number of Protons 6
Multiplicity
0.923- 0.943
Doublet
1.155- 1.176
12
Doublet
3.283- 3.326
Multiplet
3.794- 3.887
Multiplet
4.125- 4.295
Multiplet
4.652- 4.777
Septet
5.296- 5.386
Multiplet
8.017
Broad Peak
8.290
Broad Peak
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Impurity 2 Preparative isolation
Purity on HPLC
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Mass identification through Direct injection (DI)
Observations from Mass spectra
Molecular Mass
Interpretation
933
(M-H)
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H1NMR of Impurity 2
Observations from H1NMR Spectra

N N H3C CH3 N H3C N O NH O N O P O OH N O N N O P O O O O O NH O O CH3 H3C
O O CH3 O CH3
CH3
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Sr. No 1 Chemical Shift 0.908- 0.927 Number of Protons 3 Multiplicity Doublet
2 3 4 5 6 7 8 9 10 11 12 13 14
1.042- 1.062 1.157- 1.178 1.200- 1.220 3.360- 3.369 3.815- 3.866 3.934- 3.981 4.120- 4.304 4.653- 4.757 4.772- 4.862 5.298- 5.385 5.402 5.471- 5.561 8.040- 8.295
3 6 12 2 1 3 4 1 2 2 2 4 6
Doublet Doublet Doublet Multiplet Multiplet Multiplet Multiplet Multiplet Multiplet Multiplet Broadpeak Multiplet Broadpeak
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Summary and Conclusion

SUMMARY AND CONCLUSION Characterization and Impurity profile is the description of identified and unidentified impurities 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
Bibliography 1. Kasture A.V., Mahadik K.R., Wadodkar S.G., More H.N. Pharmaceutical Analysis-IIInstrumental Methods. 16 Pune, India : Nirali Prakashan, 2006. Vol. II. 2. Chatwal G R, Anand S K. Instrumental Methods of Chemical Analysis. 5. New Delhi : Himalaya Publishing House, 2007. pp. 2.624-2.629. 3. Beckett A.H., Stenlake J.B. Practical Pharmaceutical Chemistry. 4. New Delhi : CBS Publishers and Distributors, 2007. pp. 72-73 ,85,275-277, 379. Vol. 2. 4. G H. Jeffery, J. Bassett, R C Denny. Textbookfor quantitavie chemical analysis. 5. s.l. : longman scientific and technicals. 5. Snyder L R, Glajch J LKirkland J J. Practical HPLC Method Development. 2. s.l. : A Wiley-interscience Publication, 1997. 6. Scintag, Inc. Basics of X-ray difraction. 7. Pavia, lampman, Kriz. Introduction to spectroscopy. 3rd. s.l. : Brroks/cole, Thomsan learning, 2001. 8. Silverstain. Pharmaceutical analysis. 9. James, Thomas L. Fundamentals of NMR. San Francisco : Department of Pharmaceutical Chemistry, University of California, 1998. 10. Tripathi, K D. Essential of Medical Pharmacology, 6th editionPage no. 254-261. New Delhi : Jaypee brothers medical Publishers limited, 2002. 11. Development of a Validated Liquid Chromatographic method for the determination of related substance of API. Ashenafi, D. Belgium : s.n., September 2010, pp. 1708-1716. 12. Recent Trend In Impurity Profiling of Pharmaceuticals. Kavita Pilaniya, Urmila Pilaniya, Pooja Manchandani, harish Chandrawanshi, Nitin Singh, Pratistha Jain. 3, Bhopal : s.n., 2010, Journal Of Advanced Pharmaceutical Technology and Research, Vol. 1, pp. 302-310. 13. Impurity Profile: Significance in Active Pharmaceutical Ingredient. Sanjay B. Bari, Bharati R. Kadam, Yogini S. Jaiswal, Atul A. Shirkhedkar. 1, Shirpur : s.n., 2007, Eurasian Jounal of Analytical Chemistry, Vol. 2, pp. 32-52. ISBN: 1306-3057. 14. International Journal of Pharmaeuticals Sciences Review and Research. Prabu, S.Lakshmana. [ed.] T.N.K Suriyaprakash. 2, Tiruchira ppali : s.n., August 2010, Vol. 3, pp. 66-71. ISSN 0976-044X.
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15. Barar, F.S.K. Essential of pharmacotherapeutics, 5th revised edition,Page no. 340-349. New Delhi : S. Chand & Company LTD, 2009. 16. TRIPARTITE, ICH HARMONISED. Impurities in New Drug Substance Q3 (R2) step 4. 2004. 17. Isolation of Impurities by (semi)Preprative HPLC. Aranyi, Antal. pp. 240-251. 18. Wellings, Donald A. Practicle handbook of Preprative HPLC. Chennai : Charon Tec Ltd. ISBN 13: 978-1-8-56-17466-4. 19. Tenofovir: The First Nucleotide Analog for HIV-I. Evans Branch III, Melony Floyd, Marlon Honeywell. 7, July 2002, Vol. 27, pp. 359-361. 20. International pharmacopoeia. World health Organization (WHO). August- 2009. Working document QAS/09.328. 21. Characterization of impurities in the bulk drug lisinopril by liquid chromatography/ion trap spectrometry. Pei xi zhu, Dan hua wang, Cui rong son. jornal of Zhejiang University sciences B. 22. Formulaton developmet and evaluation of emtricitabine and TDF Tablets. Mankinandan M, Kannan K, Manavalan R. Jan- March 2012, International journal of drug development & Research, pp. 247- 250. 23. USFDA. Guidance of Industry, ANDAs Impurities in drug substances. 24. Identification and Characterization of Potential Impurities in Raloxifene Hydrochloride. Reguri Buchi Reddy. May 2012, Scientia Pharmaceutica, p. 605. 25. Euracian journal of Analyticcal Chemistry. Sanjay B. Bari. 2007, Vol. 2, pp. 32-40. 26. Sonal Desai, Archita Patel, SY Gabhe. 1, 2011, Indian journal of pharmaceutical sciences, Vol. 73, pp. 79- 84. 27. Separation, Identification and Structural Elucidation of a New Impurity in the Drug Substance of Amlodipine Maleate Using LC-MS/MS, NMR and IR. G V Ram Reddy. Gyeongsan 712-749 : s.n., 2010. 28. Recent advances in use of LCMS/MSfor quantitative high throuput bioanalytical support for drug discovery. Bradely L. s.l. : Bentham sciences Publishers Ltd, 2002. 29. NMR spectroscopy in Pharmacy. Ulrike Holzgrabe, Bernd W.K. Diehl, Iwona Wawer. December 1997, Journal of Pharmaceutical and biomedical analysis.
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Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Figure 1- Retention Process in Chromatography

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Figure 2- HPLC System

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Normal Phase Chromatography

Reversed Phase Chromatography

polar bonded siloxane with non-polar

Nonpolar solvents like heptane, Polar

hexane, cyclohexane, chloroform, acetonitrile, ethyl ether, dioxane

(Sometimes with additives of THF or dioxane).

Least polar components are eluted first

Most polar components are eluted first

Table 1- Types of Separations in liquid Chromatography (4) (2)

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Figure 3- X-ray diffraction Technique (6)

Figure 4- DSC Principle

Figure 5- DSC Model thermogram

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Table 2- IR regions (2)

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Incandesc ent lamp

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Figure 8- simplified chart of common functional group with characteristic absorptions

C-C C-O C-C

Stretch Stretch Stretch

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Table 3- IR positions of various bond vibrations (7)

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Figure 9- Mass Spectrometry Instrumentation

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

P; so all those which have nuclear spin quantum

O) do not have a nuclear spin because of their spin quantum no is

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

H1 H2 C13 N15 F19 P31

1/2 1 1/2 1/2 1/2 1/2

99.984 0.015 1.108 0.365 100 100

26.752 4.106 6.726 -2.710 25.167 10.829

100.000 0.965 1.590 0.104 83.300 6.630

NMR tube diameter

Min. Conc Other Nuclei 0.5mM 0.3mM

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management

1. process condition 2. analytical specification 3. long term storage condition

Maximum Daily dose 2g/day 2g/day

Qualification threshold 0.1% or 1mg/day intake 0.05%

Sobhaben Pratapbhai Patel, School Of Pharmacy & Technology Management