Thesis Final

Contents
LIST OF FIGURES .................................................................................................................. I LIST OF TABLES .................................................................................................................. II LIST OF ABBREVIATIONS .................................................................................................... III 1.0 INTRODUCTION .............................................................................................................. 1 2.0 LITERATURE SURVEY ..................................................................................................... 3 2.1 Introduction to Analytical Methods ...................................................... 3 2.2 Chromatography .................................................................................. 5 2.2.1 High Performance Liquid Chromatography ....................................... 6 2.3 Ultra violet spectroscopy.................................................................... 10 2.4 X-ray difractrometer (XRD) ............................................................... 11 2.5 Differential Scanning calorimeter (DSC)............................................ 12 2.6 Infrared spectroscopy (IR).................................................................. 14 2.7 Mass spectrometry ............................................................................. 21 2.8 Nuclear Magnetic Resonance (NMR) ................................................. 23 2.9 Impurity Profiling .............................................................................. 29 2.9.1 Origin of Impurities ........................................................................ 30 1.9.2 Need for Impurity Profiling ............................................................. 30 2.9.3 Classification of Impurities3 ............................................................ 30 2.9.4 General scheme for impurity profiling ............................................. 32 2.9.5 Impurities decision tree for Generic drug as per USFDA3 ................ 33 2.9.6 ICH decision tree for safety studies3 ................................................ 34 2.9.7 Goals for the impurity investigation ................................................ 35 2.9.8 Qualification of impurities .............................................................. 35 2.10 Drug profile ..................................................................................... 36 2.11 Review of Patents and Research articles ........................................... 38 3.0 RESEARCH ENVISAGED ................................................................................................ 41 3.1 Objectives .......................................................................................... 41 4.0 EXPERIMENTAL WORK................................................................................................. 42 4.1 List of Instruments used ..................................................................... 42 4.2 List of Chemicals ............................................................................... 43 4.1 Identification and Characterization of API ......................................... 44
4.1.1 Ultra-Violet Spectroscopy ............................................................... 44 4,1.2 Infrared Spectroscopy ..................................................................... 44 4.1.3 X-Ray Diffraction ........................................................................... 44 4.1.4 Differential Scanning Calorimeter ................................................... 44 4.1.5 Mass Spectroscopy.......................................................................... 45 4.1.6 Nuclear Magnetic Resonance .......................................................... 45
4.1.7 Development of Liquid Chromatography Method Suitable for LCMS .. 45
4.1.8 HPLC Method................................................................................. 46 4.1.9 Method for LCMS........................................................................... 47 Trial 2 ............................................................................................. 48 Trial 3 ............................................................................................. 49 Final Method................................................................................... 50 4.1.10 Mass Identification of required Peaks ............................................ 51 4.1.11 Preparative Isolation of Impurities ................................................. 51 4.1.12 NMR of impurities ........................................................................ 51 5.0 RESULTS AND DISCUSSION ........................................................................................... 53 5.1 Characterization of API...................................................................... 53 5.1.1 UV absorbance ................................................................................ 53 5.1.2 XRD (X-ray Diffraction) ................................................................. 54 5.1.3 Differential Scanning Calorimeter (DSC) ........................................ 57 5.1.4 Infrared Spectroscopy (IR) .............................................................. 58 5.1.5 Nuclear Magnetic Resonance (NMR) .............................................. 60 H1NMR.......................................................................................... 60 D- Exchange NMR......................................................................... 63 Distorytionless Enhancement by Polarization Transfer (DEPT) ...... 66 Correlation Spectroscopy (COSY)/ 2D NMR ................................. 67 5.1.6 Mass Spectrometry (MASS) of API ................................................ 68 5.2 Impurity Profiling .............................................................................. 69 5.2.1 HPLC Chromatogram ..................................................................... 69 5.2.2 LCMS Method development ........................................................... 70 5.2.3 Mass identification through X-Caliber............................................. 71 5.2.4 Preparative isolation of impurity 1 .................................................. 72 5.2.4.1 Purity on Analytical HPLC of impurity 1 ..................................... 73 5.2.4.2 Mass identification through DI (Direct Injection) of impurity 1 .... 74
5.2.5 Preparative isolation of Impurity 2 .................................................. 78 5.2.5.3 Mass identification through Direct injection (DI) of impurity 2 .... 79 5.2.5.4 H1NMR of Impurity 2 .................................................................. 80 5.2.5 Source of impurities ........................................................................ 83 6.0 SUMMARY AND CONCLUSION ............................................................................. 84 BIBLIOGRAPHY.................................................................................................................. 85
List of Figures
Figure 2.1- Separation Process in Chromatography ................................................................ 6 Figure 2.2- HPLC System ...................................................................................................... 7 Figure 2.3- X-ray diffraction Technique ............................................................................... 12 Figure 2.4- DSC Principle .................................................................................................... 13 Figure 2.5- DSC Model thermogram .................................................................................... 13 Figure 2.6- IR sources .......................................................................................................... 17 Figure 2.7- IR Detectors ....................................................................................................... 18 Figure 2.8- simplified chart of common functional group with characteristic absorptions ..... 19 Figure 2.9- Mass Spectrometry Instrumentation ................................................................... 21 Figure 2.10- The charged nucleus creates a magnetic field B and is equivalent to a small bar magnet whose axis is coincident with the spin ................................................. 23 Figure 2.11- For nuclei (I=1/2) in a magnetic field of strength Bo at thermal equilibrium ..... 25 Figure 2.12- 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 ................. 26 Figure 2.13- Mechanism of Action of API ........................................................................... 38
Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai
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List of Tables
Table 2.1- Types of Separations in liquid Chromatography .................................................... 9 Table 2.2- UV regions .......................................................................................................... 10 Table 2.3- IR regions ........................................................................................................... 14 Table 2.4- IR positions of various bond vibrations ............................................................... 19 Table 2.5- Nucleus and relative quantum activity ................................................................. 27 Table 2.6- Concentration for preparation of NMR sample .................................................... 27 Table 2.7- Goals for impurity profiling................................................................................. 35 Table 2.8- Criteria for impurity profiling .............................................................................. 35 Table 2.9- Drug profile ........................................................................................................ 36
Table 4.1- List of Instruments .............................................................................................. 42 Table 4.2- List of Chemicals ................................................................................................ 43 Table 4.3- HPLC Method ..................................................................................................... 46 Table 4.4- Method for LCMS (Trial 1) ................................................................................. 47 Table 4.5- Method for LCMS (Trial 2) ................................................................................. 48 Table 4.6- Method for LCMS (Trial 3) ................................................................................. 49 Table 4.7- Method for LCMS (Final Method) ...................................................................... 50 Table 4.8- Preparative HPLC Method .................................................................................. 50
Table 5.1- XRD observations ............................................................................................... 55 Table 5.2- IR observations ................................................................................................... 59 Table 5.3- Observations of H1NMR of API ......................................................................... 61 Table 5.4- C13 NMR observations ....................................................................................... 65 Table 5.5- Mass spectra interpretations ................................................................................ 68 Table 5.6- Mass spectra interpretation .................................................................................. 74 Table 5.7- H1 NMR Observations......................................................................................... 76 Table 5.8- interpretation of Mass spectra .............................................................................. 79 Table 5.9- H1 NMR observations ......................................................................................... 81
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List of Abbreviations
API- Active pharmaceutical ingredient LOD- Limit of detection UV- Ultraviolet HPLC- High performance liquid chromatography NPC- Normal phase chromatography RPC- Reverse phase chromatography DAD- Diode array detector XRD- X-ray diffraction DSC- Differential scanning calorimeter IR- Infrared MS- Mass TOF- Time of flight NMR- Nuclear magnetic resonance FID- Free induction decay RF- radio frequency DEPT- Distorytionless Enhancement by Polarization Transfer NOE- Nuclear overhausar effect COSY- Correlation spectroscopy FDA- Food and drug Administration ICH- International conference on harmonization USP- United states pharmacopoeia NF- National formulatory HIV- Human deficiency virus AIDS- Acquired immune deficiency syndrome TDF- Tenofovir disoproxil fumarate
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ANDA- Abbreviated new drug application ppm- parts per million GC- Gas Chromatography HPTLC- High Performance Thin Layer Chromatography
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Chapter 1
1.0 Introduction
Introduction
Drug characterization work is for evidential purpose, it is advantageous to obtain as much information on a drug sample is possible. Carrying out the characterization study, it relies mainly on chemical and physical examination. The most appropriate approach for drug characterization depends on the type of sample like tablets, capsules etc. The most simple and convenient type of investigation is a visual inspection of the physical examination of the sample. The colour, odour or general appearance of sample can be observed. And other parameters are observed for their characteristics. Other parameters like polymorphic form, functional group analysis, nuclear magnetic resonance study and their HPLC study. 1 Today a most of the drugs used are of synthetic origin. These are produced in bulk and used for their therapeutic effects in pharmaceutical formulations. 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 generally 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. Thus, the analytical activities concerning impurities in drugs and their relative amount present in drug substance are among the most important issues in modern pharmaceutical analysis. 2 According to various regulatory authorities like ICH, USFDA, MHRA etc, have imposed various guidelines regarding impurities for a drug substances. These guidelines are recommends the acceptable amounts for residual solvents in pharmaceuticals for the safety of the patient. These guidelines are recommends use of less toxic solvents and describe levels considered to be toxicologically acceptable for some residual solvents and other impurities. The guidelines are intended to provide guidance for registration applications on the content and qualification of impurities in new drug substances (NDAs) and Abbreviated new drug applications (ANDAs) produced by chemical syntheses and not previously registered in a region. It is not intended to apply to new drug and abbreviated new drug substances used during the clinical research stage of development.
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Chapter 1
Impurities in drug materials should addresses from two perspectives:
Introduction
1. Chemistry Aspects includes classification and identification of impurities, report generation, listing of impurities in specifications, and a in brief discussion of analytical procedures.3 2. Safety Aspects include specific guidance for qualifying these impurities which were not present at all, or were present at significantly lower levels, in batches of a new drug substance used in safety and clinical studies. 3 Macleods pharmaceutical Ltd. is involved in manufacturing and marketing of the various classes of therapeutic agents in global market. And so an anti retro viral drug in tablet dosage form from Macleods pharmaceutical required proper impurity profiling of the drug substance for ANDA filling. So the present research work describes the characterization and impurity profiling of the same..
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Chapter 2 2.0 Literature survey
Literature survey
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.4 Pharmaceutical analysis mainly deals with 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.5 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 various regulatory guidelines, the pharmaceutical investigation 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.4 2.1 Introduction to Analytical Methods There are various methods of analysis which can be broadly classified into two categories; Classical methods and Instrumental methodsClassical Methods5,6,7 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 gives as the basis for calculation. .
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Chapter 2
Instrumental Methods of Chemical Analysis
Literature survey
Instrumental methods are 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 regarding safety of foods, drugs, and water. Instrumental methods are widely used by Analytical scientists to utilize time smartly, and to obtain highest possible accuracy. Most instrumental techniques fit into one of the principal areas like spectroscopy, electrochemistry, chromatography and miscellaneous techniques.6 Most instrumental techniques are based one of the four-principle areas Spectrophotometric techniques UV and Visible Spectrophotometry Fluorescence Spectrophotometry 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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Chapter 2
Hyphenated Methods GC- MS (Gas chromatography - Mass Spectroscopy) LC-MS (Liquid Chromatography - Mass Spectroscopy)
Literature survey
GC- IR (Gas chromatography - Infrared Spectrophotometry) LC-IR (Liquid Chromatography - Infrared Spectrophotometry) Miscellaneous Techniques Thermal analysis Kinetic Techniques Electrophoresis
2.2 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.8 Principle 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 9
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Chapter 2
Literature survey
Figure 2. 1- Separation Process in Chromatography Source: ETS laboratories, http://www.etslabs.com 2.2.1 High Performance Liquid Chromatography 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.8 Liquid Partition Chromatography is of two types6 Normal Phase Chromatography (NPC) Reversed Phase Chromatography (RPC) 1. Normal Phase chromatography- The stationary phase is polar and mobile phase is nonpolar. 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. 2. Reverse Phase chromatography- 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.
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Chapter 2
Literature survey
Figure 2. 2- HPLC System Source: http://www.forumsci.co.il/HPLC/modes/modes1.html
Components of HPLC8 Typical HPLC instrument consists of the following main componentsA) 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 assures the constant and continuous flow of the mobile phase through the system.
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Chapter 2
C) Injector
Literature survey
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 very common detector used by the 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 more than the background (mobile phase), a positive signal is obtained.8 F) Data Acquisition and Control System Computer-based mechanism that controls all parameters of HPLC instrument like eluent composition, mixing of different solvents, temperature, injection sequence, etc and acquires data from the detector and monitors system performance.
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Chapter 2
Literature survey
Table 2 1- Table 2.1- Types of Separations in liquid Chromatography

Normal Phase Chromatography Reversed Phase Chromatography
Retention through interaction of the Retention through interaction of stationary phases polar surface the stationary phases non-polar
Mechanism
with polar parts of the sample hydrocarbon chain with non-polar molecules. parts of sample
bonded siloxane with the polar bonded siloxane with the nonfunctional group like SiO2, Al2O3, - polar functional groups like nStationary Phase
NH2, -CN, -NO2, - Diol
octadecyl (C-18) or n- octyl (C-8), ethyl, phenyl, -(CH2) n-diol, (CH2) n-CN molecules.
Nonpolar solvents like heptane, Polar solvents for e.g. methanol,

Mobile Phase
hexane, cyclohexane, chloroform, acetonitrile, Ethyl ether, dioxane
water
or
buffer
(Sometimes with additives of THF or dioxane).
Elution Order
Least polar components are eluted first
Most polar components are eluted first
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Chapter 2
2.3 Ultra violet spectroscopy
Literature survey
The wavelength range of UV radiation starts at blue end of the visible light and extended up to 2000. The ultraviolet region is subdivided into following two spectral regions: Table 2 2- UV 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.4 2.3.1 Theory of spectrophotometry4 Lamberts law: This law can be stated as follows: When a beam of light is allowed to pass through the 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 the transparent medium, the rate of increasing of concentration is directly proportional to the intensity of light.
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Chapter 2
T = C / Co A = -log T = - log (C / Co) Beers and Lamberts law
Literature survey
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. 2.4 X-ray difractrometer (XRD) About 95% of all solid materials can be classified as crystalline substance. When Xrays are interacting 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 generally employed for characterization and identification of polycrystalline substances.10 Solid matter can be described as1. Amorphous: The atoms are arranged in a random way similar to the arrangement disorder found in a liquid. Glasses are amorphous materials.
2. Crystalline: The atoms are arranged in a very regular pattern, and there is as smallest volume element that by repetition in three dimensions (X, Y, and 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 copper (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.10
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Chapter 2
Literature survey
Figure 2. 3- X-ray diffraction Technique Source: Scintag, Inc. Basics of X-ray difraction 2.5 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 is needed to flow to it than the reference to maintain both at the same temperature. In this 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 utilize for observing 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
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Chapter 2
Literature survey
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 2. 4- DSC Principle Source: http://www.evitherm.org/default.asp?lan=1&ID=982&Menu1=982
Figure 2. 5- DSC Model thermogram Source: http://www.flemingptc.co.uk
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Chapter 2
2.6 Infrared spectroscopy (IR)
Literature survey
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 to 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 have characteristic absorption frequencies known as group frequencies, which are almost independent of the nature of the rest of the molecules.11,12 Table 2 3- IR regions 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
2.6.1 Modes of vibrations In a polyatomic molecule, each atom is having three degree of freedom in three directions 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.11 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 vibrations11
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Chapter 2
Stretching vibrations
Literature survey
The atoms move essentially along the bond axis. This vibrations corresponding to the one dimensional motion, so there will be n-1 stretching vibrations for 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 group with respect to one another. E.g. twisting, rocking, torsional Types of Stretching and Bending vibrations Stretching vibrations: 1. Symmetric 2. Asymmetric
Symmetric
Asymmetric
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Chapter 2
Bending vibrations: 1. 2. 3. 4. Scissoring Rocking Twisting Wagging
Literature survey
Scissoring
Wagging
Rocking Instrumentation13 Usually optical materials, glass or quartz absorb strongly in the IR region. 14 The main parts of the IR spectrometer are as follow 1. IR radiation sources 2. Monochromators 3. Sample cells
Twisting
4. Detectors
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Chapter 2
IR radiation sources
Literature survey
Figure 2. 6- IR sources
Monochromators The radiation source emits the radiation of various frequencies. As the sample in IR spectroscopy absorbs only at some specific frequencies, it therefore becomes necessary to select desired frequencies from the radiation sources and reject the radiation of other frequencies. This selection 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.
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Chapter 2
Literature survey
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
Figure 2. 7- IR Detectors
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Chapter 2
2.6.2 Interpretation of IR spectra11,13
Literature survey
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 2. 8- simplified chart of common functional group with characteristic absorptions Source: Pavia, lampman, Kriz. Introduction to spectroscopy. 3rd. s.l. : Brroks/cole, Thomsan learning, 2001. Pg no. 13 Table 2 4- IR positions of various bond vibrations Bond C-H Mode Stretch Stretch (2v) Stretch (3v) Stretch (C) Bend in plane Bend out of plane Rocking C-C C-O Stretch Stretch Wavenumber (cm-1) 2700 - 3300 5600 6300 8300 9000 4200 5000 1300 1500 800 830 600 900 800 - 1200 900 - 1300
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Chapter 2
Bond C-C Mode Stretch
Literature survey
Wavenumber (cm-1) 800 1200 1600 1700 C=O 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 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
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Chapter 2
2.7 Mass spectrometry
Literature survey
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.9 Mass spectrometry is capable of providing information on The element composition of the sample of matter. The qualitative and quantitative composition of complex mixtures, The structure and composition of solid surfaces, Instrumentation14,15
Figure 2. 9- Mass Spectrometry Instrumentation Source: McGraw Hill online learning centre http://www.mhhe.com/physsci/chemistry/carey/student/olc/ch13ms.html 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-
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Chapter 2
Batch inlet systems, Direct probe methods, Capillary electrophoretic inlet systems. Ionic sources
Literature survey
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 (EI), Arc and spark ionization, Photo ionization, Thermal ionization, Chemical ionization. Mass analyzer9 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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Chapter 2
2.8 Nuclear Magnetic Resonance (NMR)
Literature survey
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, 13C, 15N, 19F, and 31P; 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, 14N and 16O) do not have a nuclear spin because of their spin quantum no is zero. 16
Figure 2. 10- The charged nucleus creates a magnetic field B and is equivalent to a small bar magnet whose axis is coincident with the spin Source: Fundamentals of NMR. San Francisco : Department of Pharmaceutical Chemistry, University of California, 1998.
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Chapter 2
The resonance phenomenon
Literature survey
The small nuclear magnet may spontaneously "flip'' from one orientation (energy state) to the other as the nucleus lies 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 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 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, Table lists the gyromagnetic ratios for several nuclei of interest.17
Page | 24
Chapter 2
Literature survey
Figure 2. 11- For nuclei (I=1/2) in a magnetic field of strength Bo at thermal equilibrium
Source:Fundamentals of NMR. San Francisco : Department of Pharmaceutical Chemistry, University of California, 1998. At magnetic field strengths used in NMR experiments the frequencies 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 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 fulfil the resonance condition 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 favoured 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, there is not 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
Page | 25
Chapter 2
Literature survey
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). As 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 an 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.17
Figure 2. 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
Source: Fundamentals of NMR. San Francisco : Department of Pharmaceutical Chemistry, University of California, 1998.
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Chapter 2
Table 2 5- Nucleus and relative quantum activity Nucleus Spin quantum number Natural Abundance Gyromagnetic ratio
Literature survey
Sensitivity
Electric Quadrupole 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 pharma analysis. 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).17
Table 2 6- Concentration for preparation of NMR sample

NMR tube diameter Minimum volume Min. Conc. H1 5mm 8mm 0.25ml 1ml 0.25mM 0.15mM Min. Conc. Other Nuclei 0.5mM 0.3mM
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Chapter 2
Literature survey
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 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 approximately the amount and concentration needed for structural studies on nucleic acids, polysaccharides and proteins in the size range 3-25 kilodaltons.17 2.8.1 C13 NMR 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 presses freely with their characteristic frequencies.17 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.13 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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Chapter 2
2.8.2 DEPT
Literature survey
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 expends 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 not recorded in the DEPT. but it is detected in normal C13 NMR spectra.11 2.8.3 D- Exchange NMR 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 overhausar 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.6 2.9 Impurity Profiling Today most of the drugs are synthesized chemically. They are produced in bulk and used for their therapeutic effects in pharmaceutical formulations. There are biologically active chemical compounds generally formulated into convenient dosage forms such as tablets, capsules, suspensions, ointments and injectables. These formulations deliver the drug products in a stable, non-toxic and in patient compliance 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 medicinal therapy. The safety of medicines 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 drug substances 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 normally assured by monitoring and controlling the impurities effectively. Thus, the analysis concerning impurities in drugs is among the most important issues in modern pharmaceutical analysis. 2
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Chapter 2
2.9.1 Origin of Impurities
Literature survey
Impurities in drugs are originated from various sources and phases of the synthetic process and preparation of pharmaceutical formulations. A exact difference between the process-related impurities and degradation products is always not possible. But, 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 sometimes possible that the same product of different sources may give rise to different impurities.18 1.9.2 Need for Impurity Profiling Control is more important today than ever. Until the beginning of the 20th century, drug formulations 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 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.3 2.9.3 Classification of Impurities3 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 material. They are identified or unidentified, volatile or non volatile, and it includes: Starting materials By-products Intermediates Degradation products
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Chapter 2
Reagents, ligands, and catalysts Inorganic Impurities
Literature survey
It generally derives 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. Due to these are generally known for their toxicity, the selection of appropriate controls is easily accomplished.
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Chapter 2
2.9.4 General scheme for impurity profiling
Literature survey
Page | 32
Chapter 2
2.9.5 Impurities decision tree for Generic drug as per USFDA3
Literature survey
Page | 33
Chapter 2
2.9.6 ICH decision tree for safety studies3
Literature survey
Page | 34
Chapter 2
2.9.7 Goals for the impurity investigation Table 2 7- Goals for impurity profiling Process related impurity Identify significant impurities
Literature survey
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
2.9.8 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 Quantitative Structure Activity Relationships (QSAR) database program is considered as qualified for ANDAs application. Impurities which 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 could be needed when the usual qualification threshold levels given below are exceeded.3
Table 2 8- Criteria for impurity profiling Maximum Daily dose 2g/day 2g/day Qualification threshold 0.1% or 1mg/day intake 0.05%
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Chapter 2
2.10 Drug profile
Literature survey
The Drug substance is novel reverse transcriptase inhibitor, permitted for the treatment of HIV-1 infection alone or in combination with other anti retroviral drugs. Table 2 9- Drug profile Parameters
Description
H3C CH3 O O
NH2 N
H3C
CH3
Structure
OH O O P O O HO O O
CH3
Molecular formula
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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Chapter 2
Literature survey
It inhibits the activity of HIV reverse transcriptase by competing with the natural substrate deoxyadenosine 5triphosphate and, after incorporating into DNA, by DNA chain termination. Specifically, the drug molecules are analogues of the naturally occurring deoxynucleotides required to synthesize the viral DNA and they compete with the natural
deoxynucleotides for incorporation into the growing viral DNA chain. but, unlike the natural deoxynucleotides substrates,
Mechanism of Action
NRTIs and NtRTIs (nucleoside/tide reverse transcriptase inhibitors) lack a 3'-hydroxyl group on the deoxyribose moiety. so, following incorporation of an NRTI or an NtRTI, the next incoming deoxynucleotide could not form the next 5'-3' phosphodiester bond required to extend the DNA chain. Thus, when an NRTI or NtRTI are incorporated, viral DNA synthesis is halted, this process known as chain termination. All NRTIs and NtRTIs are classified as competitive substrate inhibitors. Sever/ fatal lever problem Lactic acidosis Nausea Vomiting Pale stools Dark urine Yellowing eyes/skin Unusual tiredness
Drowsiness
Adverse effects
Precautions
Contraindicated in Hepatitis B
Uses
Treatment of HIV infection (AIDS)
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Chapter 2
Literature survey
Figure 2. 13- Mechanism of Action of API Source: http://depts.washington.eduhivaidsimagesarvresarvres_c3_d01.png
2.11 Review of Patents and Research articles 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 utilized for the characterization of
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Chapter 2
Literature survey
impurities. On the basis of mass spectral data, the structures of these impurities were identified as 3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione (impurity I), 3,7-dihydro1,3,7-trimethyl-1H-purine-2,6-dione (impurity II) and isomer of 8-chloro-1,3dimethyl-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 was employed 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 as 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 quantitative analysis 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/MS analysis. The fragmentation behavior of lisinopril and the impurities was investigated, and two unknown impurities were identified 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 for identified two 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, nearly about eight impurities were observed, and four of which were found to be new. All of the impurities were analysed 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
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Chapter 2
Literature survey
impurities, and a systematically study was carried out to characterize them. These impurities were synthesized and characterized with the help of spectral data, subjected to co-injection in HPLC, and were found to be correlated with 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)-6methyl-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 information, the structure of the unknown impurity was proposed. 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 production explaining 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.
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Chapter 3
3.0 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. 3.1 Objectives 1. API characterization using spectroscopic techniques. 2. Method development (Chromatographic parameters) 3. Impurity isolation and characterization.
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Chapter 4
4.0 Experimental Work
Experimental Work
4.1 List of Instruments used Table 4 1- 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
----
Page | 42
Chapter 4
4.2 List of Chemicals Table 4 2- List of Chemicals Chemical Name Manufacturer
Experimental Work
Grade
Acetoitrile
Rankem
HPLC
Methanol
Rankem
HPLC
Water
Milli-Q System
----
DMSO-d6
Meryer chemicals
AR
Hydrogen Peroxide
Thomas Baker
AR
Butanol
Rankem
HPLC
Trifluoroacetic Acid
Rankem
AR
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Chapter 4
4.1 Identification and Characterization of API 4.1.1 Ultra-Violet Spectroscopy
Experimental Work
Preparation of Stock Solution (1000 ppm) - 100 mg of API was weighed accurately and transferred it to 100 ml volumetric flask. 25 ml of diluents were added and sonicated it to dissolve. Volume was made up with diluent. Dilution 1 (100 ppm) - 1 ml of standard stock solution was pipetted out and transferred to 10 ml volumetric flask. Volume was made up with diluent. Final Solution (6 ppm) - 0.6 ml of above sample (Dilution 1) was pipetted out and transferred it to 10 ml volumetric flask. Volume was make up with diluent.
4,1.2 Infrared Spectroscopy Background scanning- About 10 mg of dry, finely powdered potassium bromide (IR) was triturated in mortar- pestle and spreaded it uniformly in a sample holder and compressed it with some pressure and spectra in IR Range was recorded. Sample Preparation- About 1 mg of API with approximately 300 mg of dry, finely powdered potassium bromide (IR) was Grinded the mixture thoroughly finely powdered potassium bromide IR. 4.1.3 X-Ray Diffraction Sample Preparation- The sample was loaded by back-loading method.
Scanning- Sample was 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
4.1.4 Differential Scanning Calorimeter Sample Preparation- Weighed 3-5 mg of API and transferred it to 40 l Aluminium crucible. two holes to the lid was made to escape volatile gas that evolves on thermal decomposition. The lid was crimped with crucible using crimper. Scanning- Sample was analyzed as per below mentioned parameters
Page | 44
Chapter 4
Scanning range- 35- 250 C Heating rate- 10C/ min Nitrogen Flow- 60cc/min
Experimental Work
4.1.5 Mass Spectroscopy Mode- ESI (Electron spray ionization) in Positive and Negative mode scanning 4.1.6 Nuclear Magnetic Resonance Sample Preparation- Sample was prepared using DMSO-d6 as solvent and studied with H1 NMR, C13 NMR, D-Exchange, DEPT, and COSY (Correlation Spectroscopy). 4.1.7 Development of Liquid Chromatography Method Suitable for LCMS Sample Preparation- Accurately weighed sample. Transferred it to 25 ml volumetric flask. about 15 ml of diluents were added, sonicated to dissolve and the volume was made up with diluents.
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Chapter 4
4.1.8 HPLC Method Table 4 3- HPLC Method Mobile Phase
Experimental Work
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
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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Chapter 4
4.1.9 Method for LCMS Table 4 4- Method for LCMS (Trial 1) Mobile Phase Buffer- 0.01M Ammonium Acetate pH= 6 Mobile Phase A- Buffer Mobile Phase B- Methanol
Diluents Methanol
Experimental Work
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 19 Gradient Programme 43 51 57 63 80
100 100 80 80 40 40 0 0
00 00 20 20 40 40 100 100
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Chapter 4
Trial 2 Table 4 5- Method for LCMS (Trial 2) Mobile Phase Buffer- 0.01M Ammonium Acetate pH= 6 Mobile Phase A- Buffer Mobile Phase B- Methanol : Butanol
Diluents Mobile phase
Experimental Work
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
Page | 48
Chapter 4
Trial 3 Table 4 6- Method for LCMS (Trial 3) Mobile Phase Buffer- 0.01M Ammonium Acetate pH= 6 Mobile Phase A- Buffer Mobile Phase B- Methanol : Butanol
Diluents
Experimental Work
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
Page | 49
Chapter 4
Final Method Table 4 7- Method for LCMS (Final Method) Mobile Phase Buffer- 0.01M Ammonium Acetate PH= 6 Mobile Phase A- Buffer Mobile Phase B- Methanol : Butanol
Diluents
Experimental Work
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 %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
Gradient Programme 40 45 55 60 75
Page | 50
Chapter 4
4.1.10 Mass Identification of required Peaks
Experimental Work
We were required to identify the masses of impurities which show peaks at 28.27mins and 59.34mins in LC Chromatogram and masses were identified. 4.1.11 Preparative Isolation of Impurities About 50 mg of sample was dissolved in diluents and injected it into the preparative HPLC by using manual injector. 4.1.12 NMR of impurities Impurities structure were identified by H1NMR and correlated it with obtained Mass.
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Chapter 4
Preparative HPLC Method Table 4 8- Preparative HPLC Method Mobile Phase Buffer- TFA pH= 6 Mobile Phase A- Buffer Mobile Phase B- Acetonitrile
Diluents
Experimental Work
Mobile Phase Phenyl 10.00 ml/min UV max = 260nm 100l Isocratic Time 0 10 20 %A 80 80 80 80 80 80 80 80 %B 20 20 20 20 20 20 20 20
Gradient Programme 30 40 50 60 70
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Chapter 5
5.0 Results 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 Identification of sources of impurities 5.1 Characterization of API 5.1.1 UV absorbance
UV Spectra Observation The absorbance maxima (max) is observed at the 260nm.
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Chapter 5
5.1.2 XRD (X-ray Diffraction)
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Chapter 5
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 Table 5. 1- XRD observations
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
Page | 55
Chapter 5
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
Page | 56
Chapter 5
5.1.3 Differential Scanning Calorimeter (DSC)
DSC observations 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.
Page | 57
Chapter 5
5.1.4 Infrared Spectroscopy (IR)
Page | 58
Chapter 5
IR Observation Table 5. 2- IR observations Sr. No 1 2 3 4 5 6 Wave number 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
Page | 59
Chapter 5
5.1.5 Nuclear Magnetic Resonance (NMR) H1NMR
H1NMR Observations
H3C CH3 O O
NH2 N
H3C
CH3
OH O O P O O HO O O
CH3
Page | 60
Chapter 5
Table 5. 3- Observations of H1NMR of API
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
Page | 61
Chapter 5
H1 NMR Interpretation
Page | 62
Chapter 5
D- Exchange NMR
Page | 63
Chapter 5
C13 NMR
C13NMR Observations
H3C CH3 O O
NH2 N
H3C
CH3
OH O O P O O HO O O
CH3
Page | 64
Chapter 5
Table 5. 4- C13 NMR observations
Sr. No Chemical Shift No. of Carbons
Assignments
16.890
Aliphatic -CH3 Aliphatic -CH3 Aliphatic CH2 Aliphatic CH2 Aliphatic CH
21.502
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
Page | 65
Chapter 5
Distorytionless Enhancement by Polarization Transfer (DEPT)
Observations from DEPT NMR In the API, four secondary hydrogen are present.
Page | 66
Chapter 5
Correlation Spectroscopy (COSY)/ 2D NMR
Page | 67
Chapter 5
5.1.6 Mass Spectrometry (MASS) of API
Mass Spectrometry Observations
Table 5. 5- Mass spectra interpretations

Molecular Mass (Theoretical Data) Molecular Mass (Experimental Data) Interpretation
519.4 520 (Excluding Fumaric acid Moiety) M+H
Page | 68
Chapter 5
5.2 Impurity Profiling 5.2.1 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.
Page | 69
Chapter 5
5.2.2 LCMS Method development Trial 2
Trial 3
Page | 70
Chapter 5
Final Method
5.2.3 Mass identification through X-Caliber
Page | 71
Chapter 5
5.2.4 Preparative isolation of impurity 1 Impurity 1 Trial 1
Trial 2
Page | 72
Chapter 5
5.2.4.1 Purity on Analytical HPLC of impurity 1
Page | 73
Chapter 5
5.2.4.2 Mass identification through DI (Direct Injection) of impurity 1
Observations from Mass Spectra Table 5. 6- Mass spectra interpretation

Molecular Mass (Experimental Data) Interpretation
817
(M-H)
Page | 74
Chapter 5
5.2.4.3 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
Page | 75
Chapter 5
Table 5. 7- H1 NMR Observations
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
Page | 76
Chapter 5
H1NMR Interpretation of Impurity 1
Page | 77
Chapter 5
5.2.5 Preparative isolation of Impurity 2 Preparative isolation
5.2.5.2 Purity on analytical HPLC of impurity 2
Page | 78
Chapter 5
5.2.5.3 Mass identification through Direct injection (DI) of impurity 2
Observations from Mass spectra Table 5. 8- interpretation of Mass spectra
Molecular Mass
Interpretation
933
(M-H)
Page | 79
Chapter 5
5.2.5.4 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
Page | 80
Chapter 5
Table 5. 9- H1 NMR observations
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
Page | 81
Chapter 5
H1NMR Interpretation of Impurity 1
Page | 82
Chapter 5
5.2.5 Source of impurities
In both the impurities, one molecule is main API skeleton and other molecule is monoester and they are linked through CH2 group in between their terminal NH2 roup of Adenine moiety. On storage and forced degradation these impurities cannot be formed because as additional -CH2 group is required to react with APIs Disoproxil and Isoproxil molecule (based on reaction scheme). Hence on the basis of structural evaluations, these impurities are concluded as process related impurities.
Page | 83
Chapter 6
6.0 SUMMARY AND CONCLUSION
Summary and Conclusion
The provided drug substance API-X was firstly characterized for the its absorbance maxima by UV absorbance, its polymorphic form by XRD, functional group analysis by IR spectroscopy, thermal reaction by DSC and its NMR and MASS spectrometry. Among several impurities present in the API-X, two unidentified impurities eluting near 28.124, 60.020 mins were identified by using NMR, MASS spectrometry and preparative isolation. For that initially a LC-MS method was developed by taking the analytical HPLC method as a reference as pH 6 Ammonium acetate used as a buffer and by taking buffer and methanol, butanol as mobile phase in the gradient flow. And after obtaining their Masses from LCMS data, they are isolated with preparative HPLC and the isolated impurities were confirmed by the use of NMR and MASS spectrometry. Absorbance maxima (max) for API-X was observed at the 260nm, it shows X-ray diffraction was shows major peaks at 2theta value of 4.9, 10.2, 7.7, 8.0 0.2. and its melting point was observed at 11.89C. The API-X contains the OH, -CH, -NH, -C=O, P=O groups and its NMR was recorded and structure was confirmed and it was correlated with Mass data. Two isolated impurities were studied for NMR and Mass ad their structures were identified. And then the sources for the two impurities were identified. In conclusion, the API was characterized and two impurities were confirmed with their structures and they were found to be process related impurities.
Page | 84
Chapter 7
Bibliography
Bibliography 1. Drug Characterization/ Impurity Profilling Background and concept. section, National Law enforcement authorities and drug testing laborities- Scientific. Viennea : UNITED NATIONS INTERNATIONAL DRUG CONTROL PROGRAMME. 2. 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. 3. TRIPARTITE, ICH HARMONISED. Impurities in New Drug Substance Q3 (R2) step 4. 2004. 4. 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. 5. Chrristan, Gary D. Analytical Chemistry. 6. s.l. : Wiley india Pvt Ltd, 2007. 6. Chatwal G R, Anand S K. Instrumental Methods of Chemical Analysis. 5. New Delhi : Himalaya Publishing House, 2007. pp. 2.624-2.629. 7. Kasture A.V., Mahadik K.R., Wadodkar S.G., More H.N. Pharmaceutical Analysis-IIInstrumental Methods. 16 Pune, India : Nirali Prakashan, 2006. Vol. II. 8. Snyder L R, Glajch J LKirkland J J. Practical HPLC Method Development. 2. s.l. : A Wiley-interscience Publication, 1997. 9. A., Kenneth. Textbook of pharmaceutical analysis. 3. s.l. : Willey india Pvt Ltd. 10. Scintag, Inc. Basics of X-ray difraction. 11. Pavia, lampman, Kriz. Introduction to spectroscopy. 3rd. s.l. : Brroks/cole, Thomsan learning, 2001. 12. Hsu, C. P. Sherman. Infrared Spectroscopy. Handbook of Instrumental Techniques for Analytical Chemistry. 13. Skoog, Holler, Nipman. Principle of Instrumental analysis. 14. Silverstain. Pharmaceutical analysis. 15. Ohhanesian, Lena. Handbook of Pharmaceutical analysis. Newyork : Maecel Dekker Inc, 2002. 16. Willard, Dean, Settie. Instrumental method of analysis. 7. s.l. : CBS Publishers and distributors. 17. Bill Kenyon, Robert Klenberg, Christian Starley. Nuclear Magnetic Resonance Imaging- Technology for the 21st century. Texas- USA : s.n., 1995.
Page | 85
Chapter 7
Bibliography
18. Advance approaches for the impurity profiling of pharmaceutical A review. S. J. Ingale. 7, Bhopal : s.n., July 2011, INTERNATIONAL JOURNAL OF PHARMACY & LIFE SCIENCES, Vol. 2, pp. 955-962. ISSN: 0976-7126. 19. Tripathi, K D. Essential of Medical Pharmacology, 6th editionPage no. 254-261. New Delhi : Jaypee brothers medical Publishers limited, 2002. 20. 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. 21. 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. 22. 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. 23. Barar, F.S.K. Essential of pharmacotherapeutics, 5th revised edition,Page no. 340-349. New Delhi : S. Chand & Company LTD, 2009. 24. Isolation of Impurities by (semi)Preprative HPLC. Aranyi, Antal. pp. 240-251. 25. Wellings, Donald A. Practicle handbook of Preprative HPLC. Chennai : Charon Tec Ltd. ISBN 13: 978-1-8-56-17466-4. 26. Tenofovir: The First Nucleotide Analog for HIV-I. Evans Branch III, Melony Floyd, Marlon Honeywell. 7, July 2002, Vol. 27, pp. 359-361. 27. International pharmacopoeia. World health Organization (WHO). August- 2009. Working document QAS/09.328. 28. James, Thomas L. Fundamentals of NMR. San Francisco : Department of Pharmaceutical Chemistry, University of California, 1998. 29. 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. 30. 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. 31. USFDA. Guidance of Industry, ANDAs Impurities in drug substances. 32. Identification and Characterization of Potential Impurities in Raloxifene Hydrochloride. Reguri Buchi Reddy. May 2012, Scientia Pharmaceutica, p. 605. 33. Euracian journal of Analyticcal Chemistry. Sanjay B. Bari. 2007, Vol. 2, pp. 32-40.
Page | 86
Chapter 7
Bibliography
34. Sonal Desai, Archita Patel, SY Gabhe. 1, 2011, Indian journal of pharmaceutical sciences, Vol. 73, pp. 79- 84. 35. 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. 36. Recent advances in use of LCMS/MSfor quantitative high throuput bioanalytical support for drug discovery. Bradely L. s.l. : Bentham sciences Publishers Ltd, 2002. 37. NMR spectroscopy in Pharmacy. Ulrike Holzgrabe, Bernd W.K. Diehl, Iwona Wawer. December 1997, Journal of Pharmaceutical and biomedical analysis. 38. G H. Jeffery, J. Bassett, R C Denny. Textbookfor quantitavie chemical analysis. 5. s.l. : longman scientific and technicals. 39. Braun, Robert D. Introduction to instrumental analysis. Hydrabad : Pharmamed press. 40. Taakeru Higuchi. Pharmaceutical analysis. 1. s.l. : CBS Publishers and distributors, 2005. 41. Qarrat, D C. The quantiative analysis of drug. 3. s.l. : CBS Publishers and distributors, 2008. 42. Identification and structural elucidation of a new impurity in the drug substance of Amlodipine Meleate using LC-MS/MS, NMR and IR. Gosula venkat ram reddy. Gyeonagsan : s.n., 2010, Croatica Chemica Acta. ISSN 0011-1643. 43. Scientific approches for impurity profilling in new pharmaceutical substances and its products- An overview. Vijayalakshmi R. Tamilnadu : s.n., March 2012, INTERNATIONAL JOURNAL OF PHARMACEUTICAL AND CHEMICAL SCIENCES, Vol. 1, pp. 386-390. 44. Two-Dimensional NMR Spectroscopy: Background and Overview of the Experiments. Horst Kessler. England : s.n., 1998, Angew. Chem. Int. Ed., pp. 490-536. 45. Characterization of the solid-state: spectroscopic techniques. Bugay, David E. December 2000, Advance Drug dekivery Reviews, pp. 43-60. 46. Quantitative Bioanalysis by LC-MS/MS: A Review. Sharma Devanshu, Mittal Rahul, Gupta Annu, Singh Kishan, Nair Anroop. 7, AMBALA- 133207 : s.n., JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES, Vol. 7. ISSN NO- 2230 - 7885. 47. Solid-state NMR spectroscopy in pharmaceutical research and analysis. Robert T. Berendt, Diana M. Sperger, Paul K. Isbester, Eric J. Munson. 10, Lawrence, KS 66047, USA : s.n., 2006, Trends in Analytical Chemistry, Vol. 25, pp. 977-986. 48. Recent Advances in use of LC/MS/MS for Quantitative High-Throughput Bioanalytical Support of Drug Discovery. Bradley L. Ackermann*, Michael J. Berna and Anthony T. Murphy. Indianapolis, IN 46285, USA : Bentham Science Publishers Ltd., 2002, Current Topics in Medicinal Chemistry, Vol. 2, pp. 53-66.
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Chapter 7
Bibliography
49. Krstulovic. Applications of LCMS Methodology in the Development of Pharmaceuticals. France : Sanofi-Synthlabo Recherche, 2002. 50. Hyphenated Techniques in Analytical World. Londhe S.V, Mulgund S.V, Chitre T.S, Mallade P.S. Bariwal J.B, Jain K.S. 4, Pune-411041, Maharashtra, India : The Official Publication of Association of Pharmaceutical Teachers of India, 2008, Indian Journal of Pharmaceutical Education & Research, Vol. 42, pp. 393-405. 51. Two-dimensional NMR studies of acrylate copolymers. Ajaib Singh Brar, Ashok Kumar Goyal, and Sunita Hooda. 3, New Delhi : s.n., 2009, Pure Appl. Chem, Vol. 81, pp. 389415. 52. APPLICATION OF UV-SPECTROPHOTOMETRIC METHODS FOR ESTIMATION OF TENOFOVIR DISOPROXIL FUMARATE IN TABLETS. SHIRKHEDKAR ATUL A, BHIRUD CHARUSHILA H AND SURANA SANJAY J. 1, Pak. J. Pharm. Sci, Vol. 22, pp. 27-29. 53. Measurement of Specific Heat Functions by Differential Scanning Calorimetry. O'NEIL, M. J. 10, Norwalk : The Perkin-Elmer Corp, 1966, Vol. 38. 54. Pharmaceutical Impurities: An Overview. N. Rama Rao, S. S. Mani Kiranand Prasanthi N.L. 3, 2010, Indian Journal of Pharmaceutical Education and Research, Vol. 44, pp. 301-210. 55. Identification and characterization of the new designer drug 40-methylethcathinone (4MEC) and elaboration of a novel liquid chromatographytandem mass spectrometry (LC MS/MS) screening method for seven different methcathinone analogs. Pe ter Jankovics et al. Hungary : s.n., 2011, Forensic science international, pp. 213-220.
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Contents

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Chapter 2 2.0 Literature survey

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Figure 2. 2- HPLC System Source: http://www.forumsci.co.il/HPLC/modes/modes1.html

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Table 2 1- Table 2.1- Types of Separations in liquid Chromatography

NH2, -CN, -NO2, - Diol

Nonpolar solvents like heptane, Polar solvents for e.g. methanol,

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

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Figure 2. 4- DSC Principle Source: http://www.evitherm.org/default.asp?lan=1&ID=982&Menu1=982

Figure 2. 5- DSC Model thermogram Source: http://www.flemingptc.co.uk

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Electric Quadrupole 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

Table 2 6- Concentration for preparation of NMR sample

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

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

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Freely soluble in methanol and in Dimethylformide, at 25C 2C

White crystalline powder

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Treatment of HIV infection (AIDS)

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Figure 2. 13- Mechanism of Action of API Source: http://depts.washington.eduhivaidsimagesarvresarvres_c3_d01.png

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, Mumbai

Sartorius Thermo Agilent

CPA2250 (M) Max 200 Series Xpert Pro LC-8A

XRD Preparative HPLC

Bruker Mettler Toledo