1.1.

ANALYTICAL CHEMISTRY

Analytical chemistry 1-4 was the science of making quantitative measurement. In practice, quantifying analytes in a complex becomes an excise in problem solving. To be effective and efficient, analyzing samples requires expertise in: 1. The chemistry that can occur in sample 2. Analysis and sample handling methods for a wide variety of problems (the tools of the trade) 3. Proper data analysis and record keeping. Traditionally, analytical chemistry had been split into two main types, qualitative and quantitative.

1.1.1. Types:
1.1.1.1. Qualitative Qualitative seeks to establish the presence of a given element or inorganic compound in a sample. Qualitative organic analysis seeks to establish the amount of a given element or compound in sample. 1.1.1.2. Quantitative Quantitative analysis seeks to establish the amount of a given elements or compound in a sample. Most modern analytical chemistry was categorized by two different approaches such as analytical targets or analytical methods. 1.1.1.3. By analytical targets Bioanalytical chemistry Material analysis Chemistry analysis

 Environment analysis Forensics By analytical methods Spectroscopy Mass Spectroscopy Chromatography and electrophoresis Crystallography Microscopy Electrochemistry 1.1.1.4 Techniques There were many techniques available for the analysis of materials, however; they were all based on the materials interaction with energy. This interaction permits the creation of a signal that was subsequently detected and processed for its information content. The types of analysis techniques confirm with the various types of energy.

1.1.1.4.1 Spectroscopic analysis Spectroscopy measures the interaction of the material with the electromagnetic radiation. Spectroscopy consists of many different merits such as Atomic absorption spectroscopy Atomic emission spectroscopy Ultraviolet-visible spectroscopy Infrared spectroscopy Raman spectroscopy Nuclear magnetic resonance spectroscopy Photoemission spectroscopy

1.1.1.4.2. Electrochemical analysis Electrochemistry measure the interaction of the material with an electric field.
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1.1.1.4.3. Mass analysis Mass spectrometry measures mass-to-charge ratio of molecules using electric magnetic fields. There are several ionization methods: electron impact, chemical ionization, electrospray, matrix assisted laser desorption ionization, others. Also, mass spectrometry was categorized by approaches of mass analyzers: magneticsector, quadrupole mass analyzer, quadrupole ion trap, time-of-flight, Fourier transform ion cyclotron resonance. 1.1.1.4.4. Thermal analysis Calorimetry and thermogravimetric analysis measures the interaction of a material and heat. 1.1.1.4.5. Separation science Separation processes were used to decrease the complexity of the material mixtures. The most utilized separation method was chromatography. After the material was sufficiently isolated and a signal was generated, the signal must be detected and interpreted. 1.1.1.4.6. Data acquisition and analysis Specific data acquisition and data analysis technique were required to obtain the information produced by the various techniques for the material analysis named above. Research and development in this area of analytical chemistry involves interdisciplinary efforts in physics, electronics, optics, statistics and computer science. 1.1.1.5. Hybrid techniques Combinations of the above techniques produce hybrid or hyphenated techniques.

Several examples were in popular use today and new hybrid techniques were under development.

1.1.1.5.1. Methods Analytical methods rely on scrupulous attention to cleanliness, sample preparation, accuracy and precision. A standard method for analysis of concentration involves the creation of a calibration curve. If the concentration of elements or compound in a sample is too high for the detection range of a technique, it can simply be diluted in a pure solvent. If the amount in sample was below an instruments grange of measurement, the method of addition can be used. In this method a known quantity of the elements or compound under study was added and the concentration observed in the amount actually in the sample. 1.1.1.5.2. Trends Analytical chemistry research was largely driven by performance (Sensitivity, selectivity, robustness, linear range, accuracy, precision and speed) and cost (purchase, operation, training, time and space).Effort was also put into analyzing biological system. Examples of rapidly expanding fields in this area were Genomics DNA sequencing and its related research. Genetic Finger printing and DNA microarray are very popular tools and research filed. Proteomics The analysis of protein concentrations and modifications especially in response to carious stressors, at various development stages or in various parts of the body. Metabolomics Similar to proteomics, but dealing with metabolites.

Transcriptomics mRNA and its associated field. Lipidomics Lipids and its associated field. Peptidomics Peptides and its associated field. Metabolics Similar to proteomics and metabolomics, but dealing with metal concentrations and especially with their binding to proteins and other molecules.

1.2. ANALYTICAL METHOD DEVELOPMENT[5,6]

Every day many chromatographers face the need to develop a High Perfor-mance Liquid Chromatography (HPLC) separation. A good method development strategy should require only as many experimental runs as are necessary to achieve the desired final result. Finally method development should be as simple, as possible, and it should allow the use of sophisticated tools such as computer modeling. Method development often follows the series of steps summarized below:
1. Information on sample, define separation goals

2. Need for special HPLC Proceedure, sample pretreatment edure, sample pre-treatment. 3. Choose detector and detector settings settings. 4. Choose LC method; preliminary run; estimate best separation conditions

5. Optimize separation conditions

6. Check for problems or requirement for special procedure

7a. Recover purified material

7b. Quantitative calibration

7c. Qualitative method 6

8. Validate method for release to routine laboratory

1.2.1. WHAT IS KNOWN BEFORE STARTING A METHOD DEVELOPMENT

A. Nature of the sample: Before beginning method development, there is a need to review what is known about the sample in order to define the goals of separation. Important information concerning sample composition and properties: Number of compounds present Chemical structures of compounds Molecular weights of compounds pKa values of compounds UV spectra of compounds Concentration range of compounds in samples of interest Sample solubility. The chemical composition of the sample can provide valuable clues for the best choice of initial conditions for the HPLC separation. B. Separation goals The goals of HPLC separation need to be specified clearly, which include: The use of HPLC to isolate purified sample components for spectral identification or quantitative analysis It may be necessary to separate all degradants or impurities from a product for reliable content assay or not In quantitative analysis, the required levels of accuracy and precision should known (a precision of 1 to 2% is usually achievable) Whether a single HPLC procedure is sufficient for raw material or one or more different procedures are desired for formulations.
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 When the number of samples for analysis at one times is greater than 10, a run time of less than 20 minutes often will be important. Knowledge on the desired HPLC equipment, HPLC experience and academic training do the operators have? 1.2.2. SAMPLE PRETREATMENT AND DETECTION Samples come in various forms: Solutions ready for injections Solutions that require dilution, buffering, addition of an internal standard or other volumetric manipulation Solids that must first be dissolved or extracted Samples that require sample pretreatment to remove interferences and or protect the column or equipment from damage. Direct injection of the samples is preferred for its convenience and greater precision however most samples for HPLC analysis require weighing and/or volumetric dilution before injection. Best results are often obtained when the composition of the sample solvent is close to that of the mobile phase, since this minimizes base line upset and other problems. Some samples require a partial separation (pretreatment) prior to HPLC, because it is necessary to remove interferences, concentrate sample analyte or eliminate columnkillers In many cases the development of an adequate sample pretreatment procedure can be more challenging than achieving a good HPLC separation. The samples may be of two types, regular or special. The regular samples are typical mixture of small molecules (< 2000 Da) that can be separated by normal starting conditions. Where as special samples are better separated under customized conditions given in the following table.

Table 1.1. Type of samples and their requirements Type of Sample Requirements

Inorganic ions

Detection is primary problem; use ion chromatography

Isomers

Some isomers can be separated by reversed phase HPLC and are then classified as detection is primary regulations of isomers are obtainable using either normal phase normal phase or reversed phase HPLC separations with cyclodextrin-silica columns.

Enantiomers Biological compounds

These compounds require chiral conditions for their separation. Several factors make samples of this kind special mole molecular conformation, polar functionally, and a wide range of hydrophobicity.

Macromolecules

Big molecules require column packings with large pores; (>> 10-nm diameters); in addition, biological molecule require special conditions

1.2.3. DEVELOPING THE SEPARATION A. Selecting an HPLC Method and Initial Conditions: If the HPLC is chosen for the separation, the next step is to classify the sample, as regular or special. Regular samples are typical mixtures of small molecules that can be separated using
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more or less standardized starting conditions. Special samples are usually better separated with a different column and customized conditions. Choice of the Column The selection of the column in HPLC is somewhat similar to the selection of columns in GC, in the sense that, in the adsorption and partition modes, the separation mechanism is based on inductive forces, dipole-dipole interactions and hydrogen bond formation. In case of ionexchange chromatography, the separation is based on the differences in charge, size of the ions generated by the sample molecules and the nature of ionisable group on the stationary phase. In the case of size-exclusion chromatography the selection of the column is based on the molecular weight and size of the sample components. Selection of columns based on the method is briefly summarized in the following table.

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Table 1.2. Different methods of HPLC Method /Description /Columns Reversedphase HPLC Uses water-organic mobile First choice for most samples, especially Preferred Method

phase Columns: C18 (ODS), C8, neutral or non-ioniged compounds that Phenyl, trimethylsilys (TMS), dissolve in water-organic mixtures cyano. Ion pair HjPLC Uses water-organic mobile phase, A buffer to control pH, and an Ion-pair reagent. Columns: C18, C8, Cyano Normal-phase HPLC Uses mixture of organic solvents Good second choice when reversed phase or as mobile phase Columns: ion-pair HPLC is ineffective; first choice for lipophilic samples that do not dissolve well in water-organic mixtures. Acceptable choice for ionic or ionisable compounds, especially bases or cations.

cyano, diol, amino, silica.

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The following chart show the strategy recommended for choosing the experimental conditions for the first separations.

Nature of Sample

HPLC

CE

GC

SFC

TLC

Regular

Special
Inorganic ions

Neutral

Ionic

isomers enantiomers

Exploratory run (RP)

Biological Samples isocratic Gradient Ion-pair NARP Normal Phase macromolecules proteins nucleic acids Carbohydrates Synthetic polymers peptides Carbohydrates nucleotides

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Regular samples can be further classified as neutral or ionic. Samples classified as ionic include acids, bases, amphoteric compounds, and organic salts (ionized strong acids or bases). Table 1.3. Experimental conditions for the initial separation of regular sample Separation Variable Column: Dimensions (length, ID) Stationary phase Particle size 15 x 0.46 cm C8 or C18 5 m Preferred Initial Choice

Mobile Phase: Solvents A and B %B Buffer Additives

Buffer-ACN 80-100% 25 mM potassium phosphate Do not use initially

Flow rate: Temperature: Sample Size: Volume Weight

1.5-2 ml/min 35-45o C

< 25 l < 100 g

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If the sample is neutral, buffers or additives are not required in the mobile phase. Acids or bases usually require the addition of a buffer to the mobile phase. For basic or cationic samples, less acidic reversed phase columns are recommended, and amine additives for the mobile phase may be beneficial using these conditions, the first exploratory run is carried out and then improved systematically. On the basis of the initial exploratory run, isocratic or gradient elution can be selected as most suitable. At this point it may also be apparent that typical reversed phase conditions provide insufficient sample retention, suggesting the use of either ionpair or normal phase HPLC alternatively, the sample may be strongly retained with 100% ACN as mobile phase, suggesting the use of non aqueous reversed phase chromatography or normal phase HPLC. B. Getting Started on Method Development: One approach is to use an isocratic mobile phase of some average organic solvent strength (50%). A better alternative is to use a very strong mobile phase first (80-100%) then reduce % B as necessary. The initial separation with 100% B results in rapid elution of the entire sample, but few groups will separate. Decreasing the solvent strength shows the rapid separation of all components with a much longer run time, with a broadening of latter bands and reduced retention sensitivity.

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Table 1.4.Goals that are to be achieved in method development Goal Resolution Comment Precise and rugged quantitative analysis requires that RS be greater than 1.5. Separation time Quantitation < 5-10 min is desirable for routine procedures. < 2% (ISD) for assays; < 5 % for less-demanding analysis; < 15% for trace analysis Pressure < 150 bar is desirable, < 200 bar is usually essential (new column assumed) Peak height Solvent consumption Narrow peaks are desirable for large signal/noise ratios. Minimum mobile-phase use per run is desirable.

The separation achieved in the first one or two runs usually will be less than adequate. After a few additional tries, it may be tempting to accept a marginal separation, especially if no further improvement is observed. Separation or resolution is a primary requirement in quantitative HPLC analysis. Usually, for samples containing five or fewer components, baseline resolution (RS >1.5) can be obtained easily for the bands of interest. This level of resolution favors maximum precision in reported results. Resolution usually degrades during the life of the column and can vary from day to day with minor fluctuations in separation conditions. Therefore, values of RS = 2 or greater should be the goal during method development for simple mixtures. Such resolution will favor both improved assay precision and greater method ruggedness. Some HPLC assays do not require base line separation of the compounds of interest. In such cases only enough separation of individual components is required to provide characteristic retention times for peak identification.

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The time required for a separation should be as short as possible. This assumes that the other goals of previous table have been achieved, and the total time spent on method development is reasonable. The run time goal should be compared with the 2-h setup time typically required for an HPLC procedure. Thus if only two or three samples are to be assayed at one time, a run time of 20-30 min is not excessive. When lots of 10 or more samples are to be assayed, run times of 5 to 10 min are desirable. Conditions for the final HPLC method should be selected so that the operating pressure with a new column does not exceed 170 bar (2500 psi, 17 MPa), and an upper pressure limit below 2000 psi desirable. There are two reasons for this pressure limit, despite the fact that most HPLC equipment can be operated at much higher pressures. First, during the life of a column, the back pressure may rise by a factor of as much as 2, due to the gradual plugging of the column by particulate matter. Second, at lower pressures (< 170 bar), pumps, sample valves, and especially auto samplers operate much better, seals last longer, columns tend to plug less, and system reliability is significantly improved. For these reasons, a target reassure of less than 50% of the maximum capability of the pump is desirable. When dealing with more challenging samples or if the goals of separation stringent, a large number of method developments run may be required to achieve acceptable separation. C. Repeatable Separation As the experimental runs described above are being carried out, it is important to confirm that each chromatogram can be repeated. When changing conditions (mobile phase, column, temperature) between method developments, experiments, enough time must elapse for the column to come into equilibrium with the new mobile phase and temperature. Usually, column equilibrium is achieved after passage of 10-20 column volumes of the new mobile phase through the column. However, this should be confirmed by carrying out a repeat experiment under the same conditions. When constant retention times are observed in two such back to back repeat experiments it can be assumed that the column is equilibrated and the experiments are repeatable. For reversed-phase separations, longer equilibration times can result when one of the two mobile phases being interchanged contains <10% organic.
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1.2.4. COMPLETING THE METHOD DEVELOPMENT The final procedure should meet all the goals of the method development, the method should also robust in routine operation and usable by all laboratories and personnel for which it is intended Completing the Method 1. Preliminary data to show required method performance. 2. Written assay procedure developed for use by other operators. 3. Systematic validation of method performance for more than one system or operator, using samples that cover the expected range in composition and analyte concentration; data obtained for day to day and inter laboratory operation. 4. Data obtained on expected life of column and column-to-column reproducibility. 5. Deviant results studied for possible correction of hidden problems. 6. All variables (temperature, mobile phase composition, etc.) studied for effect on separation; limits defined for these variables; remedies suggested for possible problems (poor resolution of key band pair, increased retention for last band with longer run times, etc.).

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1.3. INTRODUCTION TO HPLC [7-10] Chromatography: Chromatography is a technique by which the components in a sample, carried by the liquid or gaseous phase, are resolved by sorption-desorption steps on the stationary phase. 1.1.1 High Performance Liquid Chromatography High Performance Liquid Chromatography (HPLC) is one mode of chromato -graphy; the most widely used analytical technique HPLC utilizes a liquid mobile phase to separate the components of a mixture. These components (or analytes) are first dissolved in a solvent, and then forced to flow through a chromatographic column under a high pressure. In the column, the mixture is resolved into its components. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures. HPLC as compared with the classical technique is characterized by Small diameter(2-5 mm), reusable stainless steel columns Column packing with very small (3, 5 and 10 m) particles Relatively high inlet pressures and controlled flow of the mobile phase Precise sample introduction without the need for large samples Special continuous flow detectors capable of handling small flow rates and

detecting very small amounts Automated standardized instruments Rapid analysis High resolution

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High performance is the result of many factors: Very small particles of narrow distribution range and uniform pore size and

distribution High pressure column slurry packing techniques Accurate low volume sample injectors Sensitive low volume detectors Good pumping systems

Retention mechanism In general, HPLC is a dynamic adsorption process. Analyte molecules, while moving through the porous packing bead, tend to interact with the surface adsorption sites. Depending on the HPLC mode, the different types of the adsorption forces may be included in the retention process: Hydrophobic (non-specific) interactions are the main ones in reversed-

phase separations. Dipole-dipole (polar) interactions are dominated in normal phase mode Ionic interactions are responsible for the retention in ion-exchange chromatography. All these interactions are competitive. Analyte molecules compete with the molecule at adsorption sites. So the stronger analyte molecules interacts with the surface and the weaker the eluent interaction, the longer analyte will be retained on the surface. SEC (size-exclusion chromatography) is a special case. It is the separation of the mixture by the molecular size of its components. In this mode any positive surface interactions should be avoided. Basic principle of SEC separation is that the bigger the molecule, the less possibility for her to penetrate into the adsorbent pore space, so, the bigger the molecule the less it will be retained.

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1.1.2. TYPES OF HPLC TECHNIQUES A. Based on modes of chromatography: Reverse phase chromatography Normal phase chromatography

B. Based on principle of separation: C. Based on elution technique: Isocratic separation Gradient separation Adsorption chromatography Ion exchange chromatography Size exclusion chromatography Affinity chromatography Chiral phase chromatography

D. Based on the scale of operation: Analytical HPLC Preparative HPLC

A. Based on modes of chromatography: 1. Reverse Phase Chromatography The stationary bed is non polar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. Here the more non polar the material is, the longer it will be retained. The object was to make silica less polar or non-polar so that polar solvents can be used to separate water-soluble polar compounds. Since the ionic nature of the chemically modified silica in now reversed i.e., it is non-polar or the nature of the phase is reversed, the chromatographic separation carried out with such silica is referred to as reversed phase chromatography.
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A large number of chemically bonded silica based stationary phases are available commercially. Silica based stationary phases are still more popular in reversed phase chromatography; however other adsorbents based on polymer (styrene divinyl benzene copolymer) are slowly gaining ground. The less watersoluble compounds are better retained by the reversed phase surface. The retention time decreases in the following order: Aliphatic > induced dipoles (E.g. CCl4) > permanent dipoles (E.g. CHCl3) > weak Lewis bases (Ethers, aldehydes, ketones) > strong Lewis bases (amines ) > weak Lewis acids (alcohols, phenols) > strong Lewis acids (carboxylic acids ). Also the retention increases as the number of carbon atoms increases. As general rule the retention increases with an increase in the contact area between sample molecule and stationary phase i.e., with an increase in the number of water molecules, which are released during the adsorption of a compound. Branched chain compounds are eluted more rapidly than their corresponding normal isomers. In reversed phase system the strong attractive forces between water molecules arising from the 3-dimensional intermolecular hydrogen bonded network present in the structure of water must be distorted or disrupted when a solute is dissolved. Only higher polar or ionic solutes can interact with the water structure. Now polar solutes are squeezed out of the mobile phase and are relatively insoluble in it but with the hydrogen carbon moieties of the stationary phase. Chemically bonded octadecyl silane (ODS) and alkane with 18 carbon atoms is the most popular stationary phase used in pharmaceutical industry. Since most pharmaceutical compounds are polar and water soluble, the majority of HPLC methods used for quality assurance, decomposition studies, quantitative analysis of both bulk drugs and their formulations use ODS HPLC columns. The solvent strength in reverse phase chromatography is reversed from that of adsorption chromatography (silica gel) as stated earlier. Water interacts strongly and highly with silanol groups, so that, adsorption of sample molecules become highly restricted and they are rapidly eluted as a result. Exactly opposite applies in reversed phase system; water cannot wet the non-polar (hydrophobic) alkyl group such as C18 of ODS phase and therefore does not interact with the bonded moiety. Hence water is the weakest solvent of all and gives slowest
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elution rare. The elution time (retention time) in reversed phase chromatography increases with increasing amount of water in the mobile phase. 2. Normal phase Chromatography In normal phase chromatography the stationary phase is polar adsorbent (like silica gel or any other silica based packing) and the mobile phase is generally a mixture of non-aqueous solvents (such as n-hexane or tetra hydro furan) the separation is based on repeated adsorption desorption steps polar samples are thus retained on the polar surface of the column packing longer than less polar materials. B. Based on principle of separation: 1. Adsorption Chromatography The principle of separation is adsorption. Separation of components takes place because of the difference in affinity of compounds towards stationary phase. This principle is seen in normal phase as well as reverse phase mode, where adsorption takes place. 2. Ion Exchange Chromatography The stationary bed has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time. 3. Size Exclusion Chromatography The column is filled with material having precisely controlled pore sizes, and the sample is simply screened or filtered according its solvated molecules. Large molecules are rapidly washed through the column smaller molecules penetrate inside the porous of the packing particles and elute later.

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4. Affinity / Ion- Pair Chromatography Separation is based on a chemical interaction specific to the target species. The more popular reversed phase mode uses a buffer and an added counter-ion of opposite charge to the sample with separation being influenced by pH, ionic strength, temperature, concentration and type of organic co-solvent(s). Affinity chromato-graphy, common for macromolecules, employs a ligand (biologically active molecule bonded covalently to the solid matrix).Which interacts with its homologous antigen (analyte) as a reversible complex that can be eluted by changing buffer conditions. 5. Chiral Chromatography Separation of the enantiomers can be achieved on chiral stationary phases by formation of diastereomers via derivatizing agents or mobile phase additives on a chiral stationary phase. When used as an impurity test method, the sensitivity is enhanced if the enantiomeric impurity elutes before the enantiomeric drug. C. Based on elution technique: 1. Isocratic Separation In this technique the constant eluent composition is pumped through the column during the whole analysis. 2. Gradient Separation In this technique the eluent composition (and strength) is steadily changed during the whole analysis. D. Based on the scale of operation: 1. Analytical HPLC In this only analysis of the samples are done. Recovery of the samples for reusing is normally not done, since the samples used are very low.

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2. Preparative HPLC Where analysis of the individual fractions of pure compounds can be collected using fraction collector. The collected samples are reused.

1.1.3. INSTRUMENTATION Fig-1: HPLC Instrument

In order to realize eluent flow rates with packing in the 2 to 10 m particle sizes, which are common in modern liquid chromatography, pumping pressures of up to several thousand pounds per square inch are required. As a consequence of these high pressures, the equipment

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required for HPLC tends to be more elaborate and expensive than that encountered in other types of chromatography.

A. Stationary Phases (Adsorbents) HPLC separations are based on the surface interactions, and depend on the types of the adsorption sites (surface chemistry). Modern HPLC adsorbents are the small rigid porous particles with high surface area Main adsorbent parameters are: Particle size: 3 to 10 m Particle size distribution: As narrow as possible, usually within 10% of the mean Pore size: 70 to 300

Depending on the type of the ligand attached to the surface, the adsorbent could be normal phase (-OH-NH2), or reversed-phase (C8, C18, Phenyl), and even anion (NH4+), or cation (-COO) exchangers. B. Mobile phase (eluents) In HPLC type and composition of the mobile phase (eluent) is one of the variables influencing the separation. Despite of the large variety of solvents used in HPLC, there are several common properties: Purity Detector compatibility Solubility of the sample Low viscosity Chemical inertness Reasonable price

Each mode of HPLC has its own requirements. For normal phase mode solvents are mainly non polar, for reversed-phase eluents are usually a mixture of water with some polar organic solvent such as acetonitrile. Size-exclusion HPLC has special requirements, SEC eluents
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has to dissolve polymers, but the most important is that SEC eluent has to suppress all possible interactions of the sample molecule with the surface of the packing material. C. Mobile phase reservoir, filtering The most common type of solvent reservoir is a glass bottle. Most of the manufacturers supply these bottles with the special caps, Teflon tubing and filters to connect to the pump inlet and to the purge gas (helium) used to remove dissolved air. Helium purging and storage of the solvent under helium was found not to be sufficient for degassing of aqueous solvents. It is useful to apply a vacuum for 5-10 min. and then keep the solvent under a helium atmosphere. D. Pumps The HPLC pump is considered to be one of the most important components in a liquid chromatography system which has to provide a continuous constant flow of the eluent through the HPLC injector, column, and detector. High pressure pumps are needed to force solvents through packed stationary phase beds. However, many separation problems can be resolved with larger particle packing that requires less pressure. Flow rate stability is another important pump feature that distinguishes pumps. For most types of separation stable flow rate is not very important. However, for size exclusion chromatography the flow rate has to be extremely stable. Modern pumps have the following parameters: Flow rate range: 0.01 to 10 ml/min Flow rate stability: Not more than 1% (short term) For SEC flow rate stability should be less than 0.2% Maximum pressure: Up to 5000 psi (345 bar, 340 atm).

It is desirable to have an integrated degassing system, either helium purging, or better vacuum degassing. The two basic classifications are the constant-pressure and the constant-flow pump. The constant-pressure pump is used only for column packing. The constant-flow pump is the most widely used in all common HPLC applications.
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E. Injectors Sample introduction can be accomplished in various ways. The simplest method is to use an injection valve. In more sophisticated LC systems, automatic sampling devices are incorporated where sample introduction is done with the help of auto samplers and microprocessors. In liquid chromatography, liquid samples may be injected directly and solid samples need only be dissolved in an appropriate solvent. The solvent need not be the mobile phase, but frequently it is judiciously chosen to avoid detector interference, column/component interference, and loss in efficiency or all of these. It is always best to remove particles from the sample by filtering, or centrifuging since continuous injections of particulate material will eventually cause blockage of injection devices or columns. Injectors should provide the possibility of injecting the liquid sample within the range of 0.1 to100 ml of volume with high reproducibility and under high pressure (up to the 400 psi).They should also produce minimum band broadening and minimize possible flow disturbances. The most useful and widely used sampling device for modern LC is the micro sampling injector valve. F. Columns The heart of the system is the column. Typical analytical columns are 10, 15 and 25 cm in length and are fitted with extremely small diameter (3, 5 or 10 m) particles. The internal diameter of the columns is usually 4 or 4.6 mm; this is considered the best compromise among sample capacity, mobile phase consumption, speed and resolution. Preparative columns are of larger diameter. Packing of the column tubing with the small diameter particles requires high skill and specialized equipment. For this reason, it is generally recommended that all but the most experienced chromatographers purchase pre-packed columns, since it is difficult to match the high performance of professionally packed LC columns without a large investment in time and equipment.

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In general, LC columns are fairly durable and one can expect a long service life unless they are used in some manner which is intrinsically destructive, as for example, with highly acidic or basic eluents, or with continual injections of 'dirty' biological or crude samples. It is wise to inject some test mixture (under fixed conditions) into a column when new, and to retain the chromatogram. If questionable results are obtained later the test mixture can be injected again under specified conditions. The two chromatograms may be compared to establish whether or not the column is still used. A short guard column is introduced before the analytical column to increase the life of the analytical column by removing not only particulate matter and contaminants from the solvent but also sample components that bind irreversibly to the stationary phase. In addition, in liquidliquid chromatography, the guard column serves to saturate the mobile phase with the stationary phase so that losses of this solvent from the analytical column are minimized. The composition of the guard column packing should be closely similar to that of the analytical column; the particle size is usually larger, however, to minimize pressure drop. When the guard column has become contaminated, it is repacked or discarded and replaced with a new one of the same type. Thus, the guard column is a sacrificed to protect the more expensive analytical column. For many applications, close control of column temperature is not necessary, and columns are operated at ambient temperature. Often, however, better chromato- grams are obtained by maintaining column temperatures constant to a few tenths degree centigrade. Most modern commercial instruments are now equipped with column heaters that control column temperatures to a few tenths of a degree from near ambient to 100oC to 150oC. Columns may also be fitted with water jackets fed from a constant temperature bath to give precise temperature control. G. Detectors The function of the detector in HPLC is to monitor the mobile phase as it emerges from the column.

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1. Basic detector requirements: High sensitivity Fast response Wide linear dynamic range (this simplifies quantitation) Low dead volume (minimal peak broadening) Cell design which eliminates remixing of the separated bands Insensitivity to changes in type of solvent, flow rate, and temperature Operational simplicity and reliability It should be tune able so that detection can be optimized for different compounds It should be non-destructive.

2. Choosing a Detector: Table 1.5. Types of Detectors RI Response Sensitivity Flow sensitive Temp. sensitive Universal 4 microgram Yes Yes UV/VIS Selective 5 nanogram No No Fluor Selective 3 picogram No No MS Selective 1 picogram Yes No

3. Detector sensitivity Detector sensitivity is one of the most important properties of a LC detector. Sensitivity can be associated with the slope of the calibration curve. It is also dependent on the standard deviation of the measurements. The higher the slope of your calibration curve the higher the sensitivity of your detector for that particular component, but high fluctuations of your measurements will decrease the sensitivity.

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4. Response For mass-sensitive detectors, the response R (mV/mass/unit time) is: R = hW / sM For the concentration sensitive detector, the response R (mV/mass/unit volume) is:

Where: h = peak height (mV)

W = peak width at 0.607 of the peak height (cm) F = M = s = flow rate (ml/min) mass of solute injected chart speed (cm/min)

5. Types of Detectors Generally, there are two types of HPLC detectors, bulk property detectors and solute property detectors. Bulk property detectors: These detectors are responding to a mobile phase bulk property, such as refractive index, and dielectric constant detectors. Solute property detectors:

Solute property detectors respond to some property of the solutes, which is not exhibited by the pure mobile phase. Such as UV absorbance, fluorescence. Optical detectors are most frequently used. These detectors pass a beam of light through the flowing column effluent as it passes through a low volume (~ 10 ml) flow cell.

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The most commonly used detector in LC is the ultraviolet absorption detector. A variable wavelength detector of this type, capable of monitoring from 190 to 460-600nm, will be found suitable for the detection of the majority samples. Other types of detectors RI Refractive Index-Universal analyte detector. Solvent must remain the same throughout separation. Very temperature sensitive. Sometimes difficult to stabi-lize baseline. FD Fluorescence- Excitation wavelength generates fluorescence emission at a higher wavelength. Analytes must have fluorophore group. Can react analyte with fluorophore reagent. Very sensitive and selective. More difficult methods transfer. Results very dependent upon separation conditions. MS Mass Spec- Mass to charge ratio (m/z). Allows specific compound ID. Several types of ionization techniques: electrospray, atmospheric pressure chemical ionization, electron impact. The detector usually contains low volume cell through which the mobile phase passes carrying the sample components. H. Data systems The main goal in using electronic data systems is to increase analysis accuracy and precision, while reducing operator attention. In routine analysis, where no automation (in terms of data management or process control) is needed, a pre-programmed computing integrator may be sufficient. 1.1.4. PARAMETERS USED IN HPLC [ 5,8] A. Retention time (tR) The time it takes after sample injection for the analyte peak to reach the detector is called the retention time and is given the symbol tR OR Retention time is the difference in time between the point of injection and appearance of peak maxima.
31

 = (t2 - ta) / (t1 - ta) Where, t2 t1 ta = = = = Relative retention Retention time of the second peak measured from point of injection. Retention time of the first peak measured from point of injection. Retention time of an inert peak not retained by the column measured from point of injection

B. Retention volume Retention volume is the volume of mobile phase required to elute 50% of the component from the column. It is the product of retention time and flow rate. C. Resolution (Rs) Resolution is measure of the extent of separation of two components and the base line separation achieved. Rs = 2(t2 - t1) / (w1 + w2) Where, t1 and t2 are the retention times of the first and second adjacent bands. w1 and w2 are their baseline band widths. An alternative approach gives more reliable values of Rs band widths at half height (w1/2) are measured for bands 1 and 2, W0.5.1 and W0.5.2 then calculations of Rs using above equation (or) below equation may not be reliable when Rs is less than 1. Rs = 1.18(t2 - t1) / (W0.5.1 + W0.5.2)

32

Resolution can be estimated or measured in 3 different ways: 1. Calculations based on below e.q. Rs = 2(t2 - t1) / (w1 + w2) 2. Comparison with standard resolution curves. 3. Calculations based on the valley between the 2 bands. Resolution can be expressed in terms of three parameters (k, , and N) which are directly related to experimental conditions. Rs = 1/4( - 1) N1/2 K / (1 + k) Where, K N = = = = The average retention factor for the two bands is the column plate number is the separation factor k2 / k1: k1 and k2 are values of k for adjacent bands 1and 2.

The above equation is useful in method development because it classifies the dozen (or) so many experimental variables into 3 categories: retention (k), column (N) and selectivity ().

33

D. Capacity factor (K I )

Retention factor is related to the retention time and is a reflection of the proportion of time particular solute resides in the stationary phase as opposed to the mobile phase. Long retention times results in large values of K. The capacity factor is not the same as the available binding capacity, which refers to the mass of the solute that a specified amount of medium is capable of binding under defined conditions. The capacity factor K1can be calculated for every peak defined in a chromatogram, using the following equations. K1 = tR - t0/t0 Where, tR t0 = = Retention time of a solute peak. Column dead time or Column void time solvent peak

E. Column efficiency Two related terms are widely used as quantitative measures of chromatographic column efficiency. 1. Plate height 2. Plate count N The two are related by the equation: N = L/H The efficiency of chromatographic columns increases as the plate count becomes greater and as the plate height becomes smaller. A theoretical plate is an imaginary or hypothetical unit of a column where equilibrium has been established between stationary phase and mobile phase. N is dimensionless number and reflects the kinetics of the chromatographic retention mechanism. Efficiency depends primarily on the physical properties of the chromatographic medium together with the chromatographic column and system dimensions.
34

Theoretical plate: A theoretical plate is an imaginary or hypothetical unit of a column where distribution of solute between stationary phase and mobile phase has attained equilibrium. A theoretical plate can also be called as a functional unit of the column. The column plate number increases with several factors: 1. 2. 3. 4. 5. 6. 7. Well-packed column (column quality) Longer columns Lower flow rates Smaller column-packing particles Lower mobile phase viscosity and higher temperature Smaller sample molecules Minimum extra column effects.

Column performance can be defined in terms of values of N and band asymmetry (band shape) for a test substance run under favorable conditions. The column plate number N is defined by N = 16(tR / W)2 Manual measurement of the base line band width W may be subject to error. Therefore a more practical equation for N is N = 5.54(tR / W1/2)2 Here, tR W1/2 = = is band retention time is the band width at half height.

35

1.4. VALIDATION Analytical method validation is the process of demonstrating that analytical procedures are suitable for their intended use and provide accurate test results that evaluate a product against its defined specification and quality attributes . The U.S. Federal Register states Validation data must be available to establish that the analytical procedures used in testing meets proper standards of accuracy and reliability
[15]

any analytical test methods are expected to be used in a Quality Control environment

they require an additional degree of refinement compared to research methods [12]. The following observation will explain the relationship between validation and method development. When methods are properly developed, they readily validate. Validation is not a method development tool and it does not make a method good or efficient. Validation acceptance criteria should be based on method development experience.

36

VALIDATION OF ANALYTICAL PROCEDURES [13-17] Different Types of Validation characteristics [18]

Generalized validation process for an HPLC assay method: Validation is the process of collecting documented evidence that the method performs according to its intended purpose. 1. 4.1. Precision: The closeness of agreement between a series of measurements multiple samplings of the same homogeneous sample under prescribed condition. The precision of test method is usually expressed as the standard deviation or relative standard deviation of a series of measurements. Precision may be considered at three levels: Repeatability, Intermediate Precision and Reproducibility.

37

Acceptance Criteria: Percentage Relative standard deviation (%RSD) NMT 1 % (Instrument precision) (%RSD) NMT -2% (Intra- assay precision)

1.4.2. Accuracy [18]: The ICH guideline recommends that accuracy should be determined using a minimum of nine determinations over a minimum of three concentration levels covering the specified range (ICH, 1996). Spiked samples are prepared in triplicate at three levels over a range that covers 80 -120% of the target concentration for assay methods and over a range that covers the expected impurity content of a sample for impurity methods (Shabir, 2003). There are several methods that can be used for determining accuracy. The most common include: Analyze a sample of known concentration and compare the measurement to the true value. In this case, method accuracy is the agreement between the difference in the measured analyte concentration and the known amount of analyte added. That is the accuracy or % recovered is calculated as: Cm 100 Ct Where Cm is the measured concentration and Ct is the theoretical concentration. Accuracy has also been reported as a sample is analyzed and the measured value should ideally be identical to the true value. Accuracy is represented and determined by recovery experiments. The usual range is being 10% above or below the expected range of claim. The % recovery was calculated using the formula,
% Re covery ( a b) a bX 100

Where, a Amount of drug present in sample b Amount of standard added to the sample.

38

Acceptance Criteria: For an assay method, mean recovery will be 100% 2% at each concentration over the range of 80-120% of the target concentration. For an impurity method, mean recovery will be 0.1% absolute of the theoretical concentration or 10% relative, whichever is greater for impurities in the range of 0.1-2.5 % (V/W). 1.4.3. Detection Limit: It is lowest amount of analyte in a sample that can be detected but not necessarily quantitated under the stated experimental conditions. Following are different approaches: Visual Evaluation Method: Prepare the sample solutions with known lowest possible concentrations of analyte and establish the minimum concentration at which the analyte can be reliably detected by analyzing as per test method. Based on Signal to Noise Ratio Method: The LOD can be expressed as a concentration at specified signal-to-noise ratio obtained from samples spiked with analyte. A signal-to-noise ratio between 3:1 and 2:1 is generally considered acceptable. Based on the standard Deviation of the Response and the Slope: Prepare the blank solution as per test method and inject six times into the chromatographic system. Similarly prepare the linearity solution staring from lowest possible concentration of analyte to 150 % (or as per protocol) of target concentration and establish the linearity curve.

39

The detection limit (DL) may be expressed as:

3.3 X Standard deviation of the response of the blank () LOD = Slope The slope shall be estimated from the calibration curve of the analyte.

1.4.4. Quantitation Limit: It is lowest amount of analyte in a sample, which can be quantitatively determined with acceptable accuracy and precision. Following are different approaches: Visual Evaluation Method: Prepare the sample solutions with known lowest possible concentrations of analyte and establish the minimum concentration at which the analyte can be reliably quantified by analyzing as per test method. Based on signal to noise ratio method : The LOQ can be expressed as a concentration at specified signal-to-noise ratio obtained from samples spiked with analyte. A signal-to-noise ratio of 10:1 is generally considered acceptable. The ratio recognized by the ICH (1996) is a general rule. It has been stated that The determination of LOQ is a compromise between the concentration and the required precision and accuracy. That is, as the LOQ concentration level decreases, the precision increases. Based on the standard Deviation of the Response and the Slope: Prepare the blank solution as per test method and inject six times into the chromatographic system. Similarly prepare the linearity solution staring from lowest possible concentration of analyte to 150% (or as per protocol) of target concentration and establish the linearity curve.

40

The Quantification limit ( QL ) may be expressed as : 10 X Standard deviation of the response of the blank() LOQ = Slope The slope shall be estimated from the calibration curve of the analyte. Perform the Precision and accuracy at the level of limit of quantification by spiking LOQ concentration on placebo / Drug product / Drug substance. For detail methodology and acceptance criteria refer Precision and accuracy of method. Acceptance Criteria: In Pharmaceutical application, the LOQ is typically set at minimum 0.05% for active pharmaceutical ingredients. LOQ defined as the lowest concentration providing a RSD of 5%. test

LOQ should be at least 10% of the minimum effective concentration for clinical applications 1.4.5. Specificity: The ability to assess unequivocally the analyte in the presence of components that may be expected to present, such as impurities, degradation products and matrix components, etc. Specificity shall be demonstrated by performing Placebo / blank interference and forced degradation studies. Blank interference: Prepare blank solution as per test method and analyse as per test method. Placebo interference (In case of Drug products): Prepare the placebo solution equivalent to the test concentration (Subtract the weight of active ingredient) and analyse as per test method. Force Degradation studies: Degrade the sample forcefully under the various stress conditions like Light, heat, humidity, acid / base / water hydrolysis and oxidation and ensure the degradation from 1 % to 20 %.

41

Light: Expose the Drug product, drug substance and placebo to UV light for about 200 watt hours / square meter and the overall illumination not less than 1.2 million Lu hours
[17]

for visible light. Prepare the sample and placebo solution as per test method and

analyse. Heat: Expose the Drug product, drug substance and placebo at 105 C for about 12

hours ( For substance having low melting point below 10C of its melting point ). Prepare the sample and placebo solution as per test method and analyse. Humidity: Expose the Drug product, drug substance and placebo for about 80 % RH at about 25C for about one week. Prepare the sample and placebo solution as per test method and analyse. Acid / Base: Prepare the acid or base solution of 0.1N and reflux the sample and placebo with 50 ml of acid / base solution for about 1 hour at 60C. Neutralize the solution and dissolve the contents in diluents as per test method. Change the strength of acid and base or reflux time to ensure the desired degradation. Water: Reflux the sample / placebo with 100 ml of purified water for 12 hour at 60C. Dissolve the contents in diluents as per test method. Change the reflux time so as to ensure the desired degradation. Oxidation: Reflux for 12 hour at 60C with 1 % H2O2 or suitable oxidant. Dissolve the contents in diluents as per test method. Change the reflux time so as to ensure the desired degradation. Note: Based on the physicochemical properties and literature stress conditions can be decided.

Acceptance Criteria: Placebo / Blank should not elute at the retention time of analyte peak and known impurity peak. Peak purity of analyte peak should be confirmed. Degradation of active analyte peak should be from 1% to 20%.

42

1.4.6. Linearity and range: The linearity of an analytical method is its ability to elicit test results that are directly (or by a well defined mathematical transformation) proportional to the analyte concentration in samples within a given range. The linear range of detectability that obeys Beers law is dependent on the compound analyzed and the detector used. The working sample concentration and samples tested for accuracy should be in the linear range. The claim that the method is linear is to be justified with additional mention of zero intercept by processing data by linear least square regression. Data is processed by linear least square regression declaring the regression coefficient and b of the linear equation Y= aX + b together with the correlation coefficient of determination r. For the method to be linear the r value should be close to1. Where Y is the measured output signal, X is the concentration of sample, a is the slop, b is the intercept. The range of an analytical method is the interval between the upper and lower levels of the analyte (including these levels) that have been demonstrated to be determined with precision, accuracy and linearity using the method as written. If linearity is not meeting the acceptance criteria, establish the range of concentration in which it is linear. Acceptance criteria: Coefficient of correlation should be NLT 0.99. 1.4.7. Robustness: It is a measure of method's ability to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. For example a chromatographic method, the typical method parameters need to change deliberately and verify during method validation: Flow rate Mobile phase composition Column oven temperature pH of buffer in mobile phase Filter suitability : : : : : (+/- 0.2ml/minutes). (+/- 10% of organic phase). (+/- 5C). (+/- 0.2 units). (At least two filters).
43

For Variations: 1. System suitability should meet the acceptance criteria as per test method. 2. If system suitability doesnt meet, narrow the variation range and carryout the experiment again to meet system suitability. 1.4.8. Ruggedness: The United States of Pharmacopeia (USP) defines Ruggedness as the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of normal test conditions, such as different labs, different analysts, and different lots of reagents. Ruggedness is a measure of Reproducibility of test results under normal, expected operational conditions from laboratory to laboratory and from analyst to analyst. The following are the typical method parameters need to be tested during method validation: Analyst-to-Analyst variability. Column-to-Column variability. System-to-System variability. Different days. Different Laboratories. Stability of Solutions and mobile phase. ( At least for 48 hours )

44

Table 1.6. Method Validation Requirements for Example (ICH) [18]

Method Validation requirements Precision Assay repeatability Intermediate precision (Ruggedness) Accuracy Mean recovery per concentration Limit of detection Signal to-to-noise ratio Limit of quantification Signal to-to-noise ratio Linearity/Range Correlation coefficient y-Intercept Visual Robustness System suitability met Solution stability Specificity Resolution from main peak

Acceptance Criteria

1% RSD 2% RSD

100.0% 2.0%

3:1

10:1

>0.99 10% Linear

yes 2% change from time zero

>2 min. (retention time)

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1.5. STATISTICAL PARAMETERS

Statistics consist of a set of methods and rules for organizing and interpreting observations. The precision or reproducibility of the analytical method was determined by repeating the analysis and the following statistical parameters were calculated. 1.5.1 Mean Best estimation of the population mean mcg/ml for random samples from a population. Where x n = = = = Sum of observations Mean or arithmetic average (Ex/n) Individual observed valve Number of observation

1.5.2 Standard deviation The positive square roof of the variance S.D =

1.5.3 Relative standard deviation / Coefficient of variation Measures of the spread of data compared with the mean SD RSD = ------ x 100 Mean

46

1.5.4 Standard error SE = SD / n E n S.D = = = Sum of observations Number of observation Standard deviation

1.5.5 Correlation: (Fit of regression line)

Purpose: Measurement of the relation between two or more variables / measures how close the points were to the regression line. Correlation co-efficient can range from -1.00 + 1.00 Correlation value was denoted with the letter r n(xy) (x)( y) r= (nx2 (x)2 (ny2 (y)2

1.5.6 Regression

Purpose

1. When the concentration range was so wide that the errors, both random and systematic were not independent (which

was assumption). 2. When paring was inappropriate for other reason, notably a long time span between two analysis (sample aging,

change in laboratory conditions etc.,)

47

Regression line Y = mx + b Where, b = intercept of the line with the Y axis m = Slope (tangent)

Slope m n(xy) (x)( y) m = ------------------------n( (x2)) (x)2 (y)( (x2) (x)( xy) b= ------------------------n( (x2)) (x)2

Intercept b

48

AIM AND OBJECTIVE OF THE STUDY

Aim To develop an analytical method for dasatinib monohydrate tablets by RP HPLC and to partially validate the developed method as per ICH guidelines. Objective The scope of developing and validating an analytical method is to ensure a suitable method for a particular analyte should be more specific, accurate and precise. The main objective for that is to improve the conditions and parameters, which should be followed in the development and validation. The survey of literature reveals that good analytical methods are available for the drug dasatinib monohydrate, but the existing methods are inadequate to meet the requirements. Hence it is proposed to improve the existing methods and to develop new method for the estimation of dasatinib monohydrate in pharmaceutical dosage forms.

49

PLAN OF WORK
To obtain thorough knowledge in practical HPLC method development. A step-by-step procedure of method development to be implemented and initial chromatographic conditions for assay of dasatinib monohydrate tablets was to be established. For the initial chromatographic conditions and trials, the methods to be optimized. For the initial method, validation was to be performed by the developed RP HPLC method as per ICH guidelines.

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2.1. SELECTION OF DRUG

Dasatinib an oral anti-cancer drug in the tablet dosage form was chosen for the analytical method development and the method was validated.

2.1.1. DRUG PROFILE:

Dasatinib[21] is a 2-aminothiazole-derived inhibitor of Src family kinases. Dasatinib is an oral multi- BCR/ABL and Src family tyrosine kinase inhibitor approved for use in patient with chronic myelogenous leukaemia (CML) after imatinib treatment and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). It is being evaluated for use in numerous other cancers, including advanced prostate cancer. Structure

Systematic name N-(2-chloro-6-methylphenyl)-2-({6-[4-(2-hydroxyethyl)piperazin-1-yl]-2methylpyrimidin-4-yl}amino)-1,3-thiazole-5-carboxamide

2.1.1.1. Physical and Chemical Properties:[22]

Colour- Off-white to pale yellow Form -powder Odour-none Density ~ 1.408 g/cm3 Solubility- Soluble in Dimethyl Sulfoxide, Ethanol and Methanol Partition coefficient- log Pow ~ 4.5 (n-octanol/water)1.8 pH 7.4 Dissociation constant-pK1 = 8.8 (acidic group(s))10.28

51

Melting temperature-280-2860c Molecular Formula - C22H28ClN7O3S Molecular Weight-506.02082 [g/mol] CAS number- 863127-77-9 Storage -Store solid and solution at -20 C. Category-Anti-Cancer.

2.1.1.2. PHARMACOLOGY OF DASATINIB[23]

Mechanism of action:
Dasatinib, at nanomolar concentrations, inhibits the following kinases: BCR-ABL, SRC family (SRC, LCK, YES, FYN), c-KIT, EPHA2, and PDGFR. Based on modeling studies, dasatinib is predicted to bind to multiple conformations of the ABL kinase. Dasatinib inhibited the growth of chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) cell lines overexpressing BCR-ABL. Under the conditions of the assays, dasatinib was able to overcome imatinib resistance resulting from BCR-ABL kinase domain mutations, activation of alternate signaling pathways involving the SRC family kinases (LYN, HCK), and multi-drug resistance gene overexpression.

Pharmacokinetics:
The pharmacokinetics of dasatinib have been evaluated in healthy subjects and in patients with leukemia. Absorption Maximum plasma concentrations (Cmax) of dasatinib are observed between 0.5 and 6 hours (Tmax) following oral administration. Dasatinib exhibits dose proportional increases in AUC and linear elimination characteristics over the dose range of 15 mg to 240 mg/day. The overall mean terminal half-life of dasatinib is 35 hours.
52

Distribution In patients, dasatinib has an apparent volume of distribution of 2505 L, suggesting that the drug is extensively distributed in the extravascular space. Binding of dasatinib and its active metabolite to human plasma proteins in vitro was approximately 96% and 93%, respectively, with no concentration dependence over the range of 100500 ng/mL. Metabolism Dasatinib is extensively metabolized in humans, primarily by the cytochrome P450 enzyme 3A4. CYP3A4 was the primary enzyme responsible for the formation of the active metabolite. Flavin-containing monooxygenase3 (FMO-3) and uridine diphosphate-

glucuronosyltransferase (UGT) enzymes are also involved in the formation of dasatinib metabolites. In human liver microsomes, dasatinib was a weak time-dependent inhibitor of CYP3A4. The exposure of the active metabolite, which is equipotent to dasatinib, represents approximately 5% of the dasatinib AUC. Elimination Elimination is primarily via the feces. Following a single oral dose of [14C]-labeled dasatinib, approximately 4% and 85% of the administered radioactivity was recovered in the urine and feces, respectively, within 10 days. Unchanged dasatinib accounted for 0.1% and 19% of the administered dose in urine and feces, respectively, with the remainder of the dose being metabolites.

53

Drug-Drug Interactions:
Dasatinib is not an inducer of human CYP enzymes. It is a time-dependent inhibitor of CYP3A4 and may decrease the metabolic clearance of drugs that are primarily metabolized by CYP3A4. At clinically relevant concentrations, dasatinib does not inhibit CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, or 2E1. Drugs That May Increase Dasatinib Plasma Concentrations Drugs that inhibit dasatinib CYP3A4 are ketoconazole, itraconazole, erythromycin, clarithromycin, atazanavir, indinavir, nefazodone, nelfinavir, ritonavir, saquinavir, telithromycin may decrease metabolism and increase concentrations of dasatinib . Drugs That May Decrease Dasatinib Plasma Concentrations Drugs like antacids,,famotidine induce CYP3A4 enzyme and decrease the plasma concentration of dasatinib.

Adverse Reactions:
The most frequently reported adverse events included fluid retention events such as pleural effusion; gastrointestinal events including diarrhea, nausea, abdominal pain and vomiting; and bleeding events.

Dosage and Adminstration:

The recommended dosage of dasatinib is 140 mg/day administered orally in two divided doses (70 mg twice daily [BID]), one in the morning and one in the evening with or without a meal. Tablets should not be crushed or cut; they should be swallowed whole.

54

Side Effects:
Headache, muscle pain, tiredness, weakness, dizziness ,joint pain, pain, burning or tingling in the hands or the feet, rash, skin redness ,peeling skin, swelling, redness and pain inside the mouth, mouth sores etc..,

2.1.2 ANALYTICAL PROFILE:

55

2.2 SELECTION OF METHOD

The selection of method depends upon the nature of the sample, its molecular weight and solubility. Literature survey also helps for the selection of suitable method for the analytical method development of dasatinib in its pharmaceutical dosage form.

2.2.1. LITERATURE REVIEW:

1.Sandra Roche et al., [24] studied Development of a high-performance liquid chromatographicmass spectrometric method for the determination of cellular levels of the tyrosine kinase inhibitors lapatinib and dasatinib. His study includes Cellular samples were extracted with a tert-butyl methyl ether:acetonitrile (3:1, v/v):1 M ammonium formate pH 3.5 (8:1, v/v) mixture. Separation was achieved on a Hyperclone BDS C18 (150 mm 2.0 mm 3 m) column with isocratic elution using a mobile phase of acetonitirile10 mM ammonium formate, pH 4 (54:46, v/v), at a flow rate of 0.2 mL/min. The limit of detection and limit of quantification for lapatinib was determined to be 15 and 31 pg on column, respectively. 2. Haouala et al., [25] in the year 2009 studied Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry. His study includes Reverse-phase chromatographic separation of TKIs is obtained using a gradient elution of 20 mM ammonium formate pH 2.2 and acetonitrile containing 1% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 20 min. The method was validated according to FDA recommendations, The method is precise (inter-day CV%: 1.39.4%), accurate (9.2 to +9.9%) and sensitive (lower limits of quantification comprised between 1 and 10 ng/mL). 3 . Elisa pirro et al;[26]studied Development and validation of simple, rapid, and reliable high-performance liquid chromatography (HPLC)-UV method for quantification of major tyrosine kinase inhibitors, imatinib, dasatinib, and nilotinib, in human plasma is presented. Chromatographic separation of the drugs is achieved on an RP-C18column at flow rate of 0.9 mL/min at 35C; eluate is monitored at 267 nm. Mean intra-day and inter-day precision for all compounds are 2.5 and 13.3%; mean accuracy is 13.9%; extraction recovery ranges

56

within 40.24 and 81.81 %. Calibration curves range from 10 to 0.005 g/mL. Limits of detection are 50 ng/mL for dasatinib; limits of quantification , 100 ng/mL for dasatinib. 4. Antonio DAvolio et al,[27] studied A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of PBMC concentration of tyrosine kinase inhibitors imatinib, dasatiniband nilotinib. A simple PBMC isolation and extraction procedure were applied on 1014 mL of blood aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse phase analytical column with 25 min of analytical run, at flow rate of 0.25 mL/min. Mean intraand inter-day precision for all compounds were 8.76 and 12.20%; mean accuracy was 3.86%; extraction recovery ranged within 79 and 91%. Calibration curves ranged from 50.0 to 0.25 ng. The limit of quantification was set at 0.25 ng for all the analyzed drugs.
5. Michael T. Furlong et al;[28] studied Dasatinib (Sprycel) is a potent antitumor agent

prescribed for patients with chronic myeloid leukemia (CML). To enable reliable quantification of dasatinib and its pharmacologically active metabolites in human plasma during clinical testing, a sensitive and reliable liquid chromatographytandem mass spectrometry (LCMS/MS) method was developed and validated. Samples were prepared using solid phase extraction on Oasis HLB 96-well plates. Chromatographic separation was achieved isocratically on a Luna phenylhexyl analytical column. Analytes and the stable labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The assay was validated over a concentration range of 1.001000 ng/mL for dasatinib and its two active metabolites. Intra- and inter-assay precision values for replicate QC control samples were within 5.3% for all analytes during the assay validation. Mean QC control accuracy values were within 9.0% of nominal values for all analytes. Assay recoveries were high (>79%) . 6. Silvia De Francia et al,[29] studied A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of plasma concentration of tyrosine kinase inhibitors imatinib, dasatinib and nilotinib. A simple protein precipitation extraction procedure was applied on 250 l of plasma aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline)
57

was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse phase analytical column with 20 min of analytical run, at flow rate of 1 ml/min. Mean intraday and inter-day precision for all compounds were 4.3 and 11.4%; mean accuracy was 1.5%; extraction recovery ranged within 95 and 114%. Calibration curves ranged from 10,000 to 62.5 ng/ml. The limit of quantification was set at 62.5 ng/ml for dasatinib and nilotinib. 7. Andrea Davies et al;[30] studied A high performance liquid chromatography (HPLC)
method that separates two of the currently licenced tyrosine kinase inhibitors (TKIs); nilotinib

(AMN107, Tasigna) and imatinib (STI571, Glivec), together with its main metabolite, CGP-74588, from human plasma. After solid phase extraction the drug mix was separated through a Gemini C6-phenyl column (150 mm 4.6 mm, i.d.; 5 m) (Phenomenex, UK) under isocratic mobile phase conditions of methanol:50 mM ammonium acetate (pH 8) (65:35, v/v) with ultra-violet (UV) detection at 260 nm wavelength. For all compounds the intra-day coefficient of variation and bias were <3% and <5% respectively; and inter-day were <4% and <9%. 8. John Araujo et al.,[31] studied SRC is a tyrosine kinase that plays a role in oncogenic, invasive and bone-metastatic processes. It has therefore been prioritized as a candidate therapeutic target in patients with solid tumors. Several SRC inhibitors are now in development, of which dasatinib has been most explored. Preclinical studies in a wide variety of solid tumor cell lines, including prostate, breast and glioma, have shown that that dasatinib acts as a cytostatic agent, inhibiting the processes of cell proliferation, invasion and metastasis. Dasatinib also inhibits the activity of osteoclasts, which have a major role in the development of metastatic bone lesions. Dasatinib has additive or synergistic activity in combination with a number of other agents, including cytotoxic agents and targeted therapies, providing a rationale for combination treatment in a clinical setting. Emerging clinical data with dasatinib support experimental observations, with preliminary phase 1 and 2 data demonstrating activity, both as a single agent and as combination therapy, in a range of solid tumors. Future clinical trials will further assess the clinical value of SRC inhibition with dasatinib.

58

9. D.V.Mhaske et al;[32] studied Two sensitive and reproducible methods are described for the quantitative determination of dasatinib in the presence of its degradation products. The first method was based on high performance thin layer chromatography (HPTLC) followed by densitometric measurements of their spots at 280 nm. The separation was on HPTLC aluminium sheets of silica gel 60 F254 using toluene:chloroform (7.0:3.0, v/v). This system was found to give compact spots for dasatinib after development (R F value of 0.23 0.02). The second method was based on high performance liquid chromatography (HPLC) of the drug from its degradation products on reversed phase, PerfectSil column [C18 (5 m, 25 cm 4.6 mm, i.d.)] at ambient temperature using mobile phase consisting of methanol:20 mM ammonium acetate with acetic acid (45:55, v/v) pH 3.0 and retention time (t R = 8.23 0.02 min). Both separation methods were validated as per the ICH guidelines. No chromatographic interference from the tablet excipients was found. Dasatinib was subjected to acidalkali hydrolysis, oxidation, dry heat, wet heat and photo-degradation. The drug was susceptible to acidalkali hydrolysis and oxidation. The drug was found to be stable in neutral, wet heat, dry heat and photo-degradation conditions. As the proposed analytical methods could effectively separate the drug from its degradation products, they can be employed as stability indicating. 10. Eva Karlj et al;[33] studied Imatinib, dasatinib and nilotinib are three tyrosine kinase inhibitors currently used to treat Bcr-Abl1 positive chronic myelogenous leukaemia (CML). After the addition of isotopically labelled internal standard, the drug was extracted with 0.1% formic acid in methanol. The collected extract (1 L) was injected onto a Phenomenex Kinetex 50 mm 2.1 mm C18 column and eluted with acetonitrile gradient into a triple quadrupole ESIMS/MS Agilent 6460 operated in positive mode. The total run time was only 2.6 min. The method was validated in terms of linearity, selectivity, specificity, accuracy, precision, absolute and relative matrix effect and stability. The effect of haematocrit (Hct) on the accurate concentration determination was also examined. The method was linear in the range of 505000 g/L for imatinib and nilotinib and in the range of 2.5250 g/L for dasatinib, with correlation coefficient values higher than 0.997. Lower limits of quantification (LLOQ) were 50 g/L for imatinib and nilotinib and 2.5 g/L for dasatinib. The method proved to be accurate (% bias < 13.2) and precise (CV < 10.3%) on intra- as well as on inter-day basis.
59

11. Lutz Gtze et al;[34] studied A simultaneous test for six TKIs (erlotinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib) was developed using liquid chromatography tandem mass spectrometry in a multiple reaction monitoring mode. After protein precipitation the specimens were applied to the HPLC system and separated using a gradient of acetonitrile containing 1% formic acid with 10 mM ammoniumformiate on an analytic RP-C18 column.The calibration range was 101000 ng/mL for sunitinib and 505000 ng/mL for the other TKIs with coefficients of determination 0.99 for all analytes. The intra- and inter day coefficients of variation were 15% and the chromatographic run time was 12 min. 12. K. Micova et al;[35] studied Therapeutic drug monitoring is recommended for the optimal several malignant diseases. The aim of this study was to develop and validate an isotope dilution direct injection mass spectrometry method for the high throughput determination of tyrosine kinase inhibitors in plasma from leukemic and cancer patients. The plasma for analysis was deproteinated by methanol and the centrifuged supernatant was directly injected to mass spectrometer without separation step. We developed a fast method with analysis time of 55 s and 19 s in multiple injection setting. The method was successfully validated and applied to the patient plasma samples. In order to overcome insufficient sensitivity of dasatinib, multiple reaction monitoring cube mode in linear ion trap (MRM3) was successfully applied. The limits of quantification were in the range 1.05.5 ng/ml. Imprecisions were lower than 6.9% and the accuracy of the quality control samples ranged between 99.0 and 107.9%. 13. Stephane Bouchate et al;[36] Tyrosine Kinase Inhibitors (TKIs) are a class of targeted drugs for the treatment of malignant pathologies. Chromatography was performed on a Waters Acquity-UPLC system with BEH C18-50*2.1 mm column, under a gradient of ammonium formateacetonitrile. An Acquity-TQD with electrospray ionization was used for detection. Samples were prepared by solid phase extraction (Oasis MCX Elution) and eluate was injected in the system.Calibration curves ranged from 10 to 5000 ng/mL for imatinib, its metabolite, nilotinib, lapatinib, erlotinib and sorafenib and from 0.1 to 200 ng/mL for dasatinib, axitinib, gefitinib and sunitinib. Peaks of each compound (retention time from 0.76 to 2.51 min) were adequately separated. The mean relative extraction recovery was in the range of 90.3106.5%.
60

14. Zhongzhou Shen et al;[37] To characterize and enable efficient rat pharmacokinetic (PK) screening in early drug discovery, automated sampling of blood time points are routinely employed. With thedevelopment of dried blood spot (DBS) technology for drug level quantitation, an opportunity exists for the automated collection of rat PK time points using DBS. DBS, as an alternative sample collection technique has led to the increased collection of PK study samples for the quantitative analyses of drug candidates in both pre-clinical and clinical studies. However, the feasibility of using DBS samples for drug metabolite profiling including both phase I and phase II metabolites has not been well established. This work reports the study of metabolite profiling of dasatinibdosed to Wistar Han rats using automated DBS collection. Automated DBS and plasma collection using a rat AccuSampler (VeruTech AB, Sweden) was employed using dasatinib as a model compound. The DBS and plasma samples were extracted by methanol and acetonitrile and both plasma and DBS extracts were analyzed using a Sciex API4000 Qtrap mass spectrometer coupled to a Shimazdzu HPLC system. Dasatinib and its metabolites were analyzed by multiple reaction monitoring (MRM) and MRM trigger enhanced product ion scan (MRM-EPI). Both phase I oxidative metabolites and phase II glucuronide conjugates and sulfate conjugates were detected from both rat plasma and DBS samples. Overall, comparable metabolite profiles including phase I oxidative and phase II glucuronide and sulfate conjugates were observed from both extracts of plasma and DBS samples when using the untreated DBS cards for dasatinib. Chemically treated DBS cards such as DMPK-A and DMPK-B cards may affect the dasatinibmetabolites. Similar PK parameters were obtained for dasatinib from both plasma and DBS samples, after correcting for blood to plasma ratio. The results obtained from this study suggest that collection of study samples by DBS can be used for metabolite profiling, however, the availability of limited samples may be a concern for multiple injections.

On literature survey it was found that, various analytical strategies have been used for the measurement of Dasatinib either alone or in combination with various drugs in plasma and pharmaceutical preparation using few spectrophotometric, High-performance liquid

chromatography (HPLC) and Reverse phase-high performance liquid chromatography (RPHPLC), LC-MS, HPLC-MS/MS, HPTLC, and LC-tandem mass spectrometry method.

61

In view of the need for a suitable method for routine analysis in formulation, attempts are being made to develop and validate simple, precise and accurate analytical methods for the estimation of Dasatinib and extend it for their determination in formulation. As chromatographic method of analysis is a pre-requisite for the marketing of most of the formulation, one HPLC and spectrophotometric methods were developed and validated for the determination of title drug. The utility of the developed method to determine the content of drug in commercial tablet is also demonstrated. Validation of the method was done in accordance with USP and ICH guideline for the assay of active ingredients. The methods were validated for parameters like accuracy, linearity, precision, specificity, and system suitability. These methods provide means to simultaneously characterize and quantify the components of a mixture without prior separation and derivatization. These proposed methods are suitable for the analysis in pharmaceutical quality control laboratories.

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2.3. SELECTION OF INSTRUMENT

Dasatinib is polar in nature the RP-HPLC method was preferred because of its simplicity and suitability. The reasons for developing RP-HPLC method for determination of the drug in tablet dosage forms are as follows: 1. 2. 3. To develop newer RP-HPLC Method by Isocratic Mode. To reduce the run time as compared with previously reported literature. To develop a method for drug in its dosage form.

Advantages of less run time in HPLC: Its beneficial to the company economically. To estimate the different compounds with less time in different formulations like tablets, capsules, syrups, expectorants and injections. Utilisation of minimum solvent. Reduce the cost. Less utilisation of men, machine and materials.

As RP-HPLC was chosen as the instrument wide variety of equipment, coloumns, eluent and operational parameters are involved in it.

2.3.1. Coloumns:
The column was one of the most important components of the HPLC because the separation of the sample components was achieved when those components pass through the column. Trials were done on different coloumns and the optimized one is Cosmicil BDS coloumn. Cosmicil BDS coloumn:[37] BDS is a base deactivated silanol in which the residual silanol groups are deactivated which is suitable for the basic,acidic and neutral analytes.

63

This

coloumn

is

suitable

for

elution hydrogen

with

different

eluents

like

methanol,acetonitrile,water,disodium phosphate, acetic acid etc.,

phosphate,potassium

dihydrogen

These coloumns shows wide pH range i.e. 2-9. The flow-rate is 0.8-2.0ml/min. The UV-detection range is 220-330nm. Column Dimensions

Type

Length (mm) 150

Width (mm) 4.6

Particle (m) 5

Size

C-18 BDS

2.3.2. Mobile Phase:

Mobile phases used for HPLC are typically mixtures of organic solvents and waters or aqueous buffers. These are chosen based on the following points: 1. The drug must be stable in moile phase for atleast duration of analysis. 2 .Excesive salt concentration must be avoided which otherwise lead to damage of the equipment. 3. Minimize the absorbance of buffer. Considering the above points methanol and acetonitrile are used as mobile phase . 2.3.2.1. Methanol:[38] It is also known as methyl alcohol. It is a polar mobile phase which is used for RP-HPLC. The UV-cutoff range 205nm.

64

Toxicity: Methanol has a high toxicity in humans. If ingested, for example, as little as 10 mL of pure methanol can cause permanent blindness by destruction of the optic nerve, and 30 mL is potentially fatal, although the median lethal dose is typically 100 mL (4 fl oz) (i.e. 12 mL/kg of pure methanol. Toxic effects take hours to start, and effective antidotes can often prevent permanent damage. Because of its similarities in both appearance and odor to ethanol (the alcohol in beverages), it is difficult to differentiate between the two (such is also the case with denatured alcohol). Methanol is toxic by two mechanisms. First, methanol (whether it enters the body by ingestion, inhalation, or absorption through theskin) can be fatal due to its CNS depressant properties in the same manner as ethanol poisoning. Second, in a process of toxication, it is metabolized to formic acid (which is present as the formate ion) via formaldehyde in a process initiated by the enzyme alcohol dehydrogenase in the liver. Methanol is converted to formaldehyde via alcohol dehydrogenase (ADH) and formaldehyde is converted to formic acid (formate) via aldehyde dehydrogenase (ALDH). The conversion to formate via ALDH proceeds completely, with no detectable formaldehyde remaining. Formate is toxic because it inhibits mitochondrial cytochrome c oxidase, causing the symptoms of hypoxia at the cellular level, and also causing metabolic acidosis, among a variety of other metabolic disturbances.

2.3.2.2. Acetonitrile:[39] It is also known as methyl cyanide Its low viscosity and low chemical reactivity make it a popular choice for liquid chromatography.

65

Toxicity: Acetonitrile has only a modest toxicity in small doses. It can be metabolised to produce hydrogen cyanide, which is the source of the observed toxic effects. Generally the onset of toxic effects is delayed, due to the time required for the body to metabolize acetonitrile to cyanide (generally about 212 hours). Acetonitrile poisoning in humans (or, to be more specific, of cyanide poisoning after exposure to acetonitrile) are rare but not unknown, by inhalation, ingestion and (possibly) by skin absorption.[13] The symptoms, which do not usually appear for several hours after the exposure, include breathing difficulties, slow pulse rate, nausea, and vomiting: Convulsions and coma can occur in serious cases, followed by death from respiratory failure. The treatment is as for cyanide poisoning, with oxygen, sodium nitrite, and sodium thiosulfate among the most commonly-used remedies.

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3.1. CHEMICALS AND INSTRUMENTS

3.1.1 MATERIALS:

A. Chemicals used: S. No. 1. 2. 3. 4. 5. Chemical name Acetonitrile Methanol Purfied Water Triethylamine Orthophosporic acid Grade HPLC HPLC MilliQ HPLC HPLC HPLC

B. Drug: S. No. 1. 2. Drug name Dasatini b(std) Dasatini b(sample) Manufactured by NATCO Pharma Pvt. Ltd. NATCO Pharma Pvt. Ltd.

67

3.1.2. Instruments used S. No. 1. Name of the instrument HPLC Column Waters Cosmicsil BDS C-18,5microns, (150X4.6mm) 2. 3. 4. 5. 6. Orbital shaker Analytical balance Membrane filter pH meter Centrifuge apparatus Afcoset Smart Labtech Pvt. Ltd., BV - 40 Make

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3.2. MATERIALS AND METHODS

3.2.1. Buffer preparation: Add 4.0ml of triethylamine to 100ml water and adjut the pH to 6.50.05 diluted with orthophosphoric acid add 10ml of methanol and mix well. 3.2.2. Solvent mixture: Prepare a mixture of methanol and acetonitrile in the ratio of 50:50 v/v respectively.

3.2.3. Mobile phase: Prepare a filtered and degassed mixture of buffer and solvent in the ratio of 50:50 v/v respectively. 3.2.4. Preparation of standard solution: Accurately weighed quantity of the drug was transfered about 68.0 mg of Dasatinib monohydrate (working standard) into 50ml volumetric flask . Add about 30ml of solvent mixture and sonicate to dissolve. Cool the solution to room temperature and dilute to volume with solvent mixture. Transfer 1.0ml of the above solution into a 10ml volumetric flask and dilute to volume with mobile phase. 3.2.5. Preparation of working standard solution: From the standard stock solution, 2.0 ml, 3.0 ml ,4.0 ml, 5.0 ml and 6.0 ml was transferred into a 10 ml volumetric flask and made up to the mark to produce 20,30,40,50,60 g/ml respectively with mobile phase.

69

3.2.6. Preparation of sample solution: Weigh and finely powder not fewer than 10 tablets. Transfer an accurately weighed portion of powder, equivalent to 68.0mg of Dasatinib into a 100ml volumetric flask. Add about 60ml of solvent mixture , shake on orbital shaker for 15min and sonicate for 30min with occasional shakings. Cool the solution to room temperature and dilute to volume with solvent mixture. Centrifuge the solution at 3000RPM for 15min. Transfer 1ml of the above solution into a 10ml volumetric flask, dilute to volume with mobile phase. 3.2.7. Preparation of Placebo The amount of powdered inactive ingredient supposed to be present in 10 tablets was accurately weighed and transferred in to 100 ml volumetric flask. Add about 60ml of solvent mixture , shake on orbital shaker for 15min and sonicate for 30min with occasional shakings. Cool the solution to room temperature and dilute to volume with solvent mixture. Centrifuge the solution at 3000RPM for 15min. Transfer 1ml of the above solution into a 100ml volumetric flask, dilute to volume with mobile phase.

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3.3. METHOD DEVELOPMENT

The objective of this study was to optimize the assay method for estimation of Dasatinib based on the literature survey made.

Trial-1
Chromatographic conditions Flow rate Column Detector wavelength Column temperature Injection volume Run time : 1.2 ml / min

: Devosil ODS C18, 150 mm X 4.6 mm, 5 m : : : : 315 nm 35 0C 10 l 10 min

Mobile phase Solution of phosphate buffer (pH-6.5), acetonitrile and methanol in the ratio of 50:50 v/v is used. Mobile phase was pumped at a flow rate of 1.2ml/min. Observation The tailing factor of the peaks obtained was high & fails the system suitability. The chromatogram for trial-1 was shown in .

71

Fig:2 Trial -1 chromatograph

Trail-2
Chromatographic conditions Flow rate Column Detector wavelength Column temperature Injection volume Run time : 1.2 ml / min

: Cosmicsil ODS C18, 150 mm X 4.6 mm, 5 m : : : : 311 nm 35 0C 10 l 10 min

Mobile phase Solution of phosphate buffer (pH-6.5), acetonitrile and methanol in the ratio of 60:40v/v at a flow rate of 1.0ml/min. Cosmicsil ODS C-18, 150 mm X 4.6 mm, 5 m column was used. The column temperature was maintained at 35 c.
72

Observation The tailing factor of the peaks obtained was high & fails the system suitability. The chromatogram for trial-2 was shown. Fig:3 Trial -2 chromatograph

Trial -3
Chromatographic conditions Flow rate Column Detector wavelength Column temperature Injection volume Run time : : : : : : 1.0 ml / min Cosmicsil BDS, C18, 150 mm X 4.6 mm, 5 m 315 nm 25 0C 10 l 10 min
73

Mobile phase Solution of phosphate buffer (pH-6.5), acetonitrile and methanol in the ratio of 50:50v/v at a flow rate of 1.0ml/min. Cosmicsil BDS C-18, 150 mm X 4.6 mm, 5 m column was used. The column temperature was maintained at 25 c. Observation Peak of Dasatinib was well resolved with the retention time of 6.46 min. The chromatogram for trial -3 (optimized method) was shown. Fig:4 Trial-3 chromatograph

3.3.1. OPTIMIZED METHOD FOR ASSAY Buffer preparation: Add 4.0ml of triethylamine to 100ml water and adjut the pH to 6.50.05 diluted with orthophosphoric acid add 10ml of methanol and mix well. Solvent mixture: Prepare a mixture of methanol and acetonitrile in the ratio of 50:50 v/v respectively.

74

Mobile phase: Prepare a filtered and degassed mixture of buffer and solvent in the ratio of 50:50 v/v respectively. Preparation of standard solution: Accurately weighed quantity of the drug was transfered about 68.0 mg of Dasatinib monohydrate (working standard) into 50ml volumetric flask .Add about 30ml of solvent mixture and sonicate to dissolve.Cool the solution to room temperature and dilute to volume with solvent mixture.Transfer 1.0ml of the above solution into a 10ml volumetric flask and dilute to volume with mobile phase. Preparation of working standard solution: From the standard stock solution, 2.0 ml, 3.0 ml ,4.0 ml, 5.0 ml and 6.0 ml was transferred into a 10 ml volumetric flask and made up to the mark to produce 20,30,40,50,60 g/ml respectively with mobile phase. Preparation of sample solution: Weigh and finely powder not fewer than 10 tablets. Transfer an accurately weighed portion of powder, equivalent to 68.0mg of Dasatinib into a 100ml volumetric flask. Add about 60ml of solvent mixture , shake on orbital shaker for 15min and sonicate for 30min with occasional shakings. Cool the solution to room temperature and dilute to volume with solvent mixture. Centrifuge the solution at 3000RPM for 15min.Transfer 1ml of the above solution into a 10ml volumetric flask, dilute to volume with mobile phase.

75

Preparation of Placebo The amount of powdered inactive ingredient supposed to be present in 10 tablets was accurately weighed and transferred in to 100 ml volumetric flask. Add about 60ml of solvent mixture , shake on orbital shaker for 15min and sonicate for 30min with occasional shakings. Cool the solution to room temperature and dilute to volume with solvent mixture. Centrifuge the solution at 3000RPM for 15min.Transfer 1ml of the above solution into a 100ml volumetric flask, dilute to volume with mobile phase. Test Procedure 10 l of the standard, sample, blank and placebo preparations in duplicate were injected separately into HPLC system and the peak responses for Dasatinib were measured. The quantities from the peak area in mg of Dasatinib were calculated per tablet taken.

Optimized Chromatographic conditions Flow rate Column Detector wavelength Column temperature Injection volume Run time Retention time Observation: The peak shape of Dasatini b was good and also optimum plate count and tailing. : : : : : : : 1.0 ml / min Cosmicsil BDS, C18, 150 mm X 4.6 mm, 5 m 315 nm 25 0C 10 l 10 min 6.467

76

Conclusion: Hence this method was finalized for the estimation of Dasatinib Calculation: The amount of drug was calculated by using the following formula: AT Assay % = WS DT P Avg. Wt

-------------- x ----------x --------- x ----------x------------------ X 100 AS DS WT 100 LC

Where
At

= = = = = = = =

Average area of sample Average area of standard Weight of standard Dilution factor of standard Dilution factor of sample Weight of sample Purity of working standard used Average weight of tablets taken for analysis

As Ws Ds Dt Wt P Aw

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Table 3.1. Assay data by HPLC

DASATINIB Standard Area 1 2 3 4 5 6 Average Sample area 1 2 3 4 5 6 Average Tablet average weight Standard weight std.purity Sample weight Label amount %Assay 311353 311363 311343 311323 311345 311333 311343.33 311297 311287 311277 311307 311267 311259 311282.33 0.04889 50 99.8 489.6 50 99.8

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3.4. VALIDATION PARAMETERS

Validation of analytical method was a process of establishing documental evidence which provides a high of assurance that a specific process will consistently produce a product of predetermined specifications and quantity attributes. The following parameters have been validated. 1. System suitability 2. Linearity 3. Accuracy 4. Precision 5. Robustness 6. LOD & LOQ

3.4.1. System Suitability: Chromatograph the standard preparations (six replicate injections) and measure the peak area responses for the analyte peak and evaluate the system suitability parameters as directed. Table 3.2. System suitability data by HPLC System suitability Parameters %RSD Tailing factor No. of theoretical plates Mirtazapine 0.78 % 1.24 7620

79

Acceptance Criteria: The number of theoretical plates for Dasatinib peak should be NLT 2000. % RSD for six replicate injectionsof peak area response for Dasatinib peak from the standard preparation should not be morethan 2.0. The tailing factor for Dasatinib should not be morethan 2.0.

From the system suitability studies it was observed that all the parameters were within limit.

3.4.2. Linearity: Linearity of the proposed HPLC method for determination of Dasatinib were evaluated by analysing a series of different concentrations of standard drug. In this study Six concentrations were chosen ranging between 20-60g mL-1 for Dasatinib. Each concentration was injected six times and obtained information on variation in the peak area response of pure analyte was plotted against corresponding concentrations and result was shown in Table . The linearity of the calibration graph was validated by the high value of correlation coefficient, slope and the intercept value was shown

80

Table:3.3. Linearity range and average area values Solution No. 1 2 3 4 5 Concentration (g / ml) 20 30 40 50 60 Peak area* 153482 228347 311353 388054 460767

*- average of 6 replicate injections for each concentration

500000 450000 400000 350000 300000 250000 200000 150000 100000 50000 0 0 10 20 30 40 50 60 70 Y=7985X-11196 R2=0.999

Calibration curve of Dasatinib Acceptance criteria Correlation coefficient should be not less than 0.999.

81

Table 3.4. Calibration parameters for Dasatinib

Parameter

Results

Slope Intercept Correlation co-efficient Percentage curve fitting

7985 -11196 0.999 99.9%

Observation The linearity Correlation coefficient for dasatinib is 0.999

3.4.3. Precision Precision of the analytical method was studied by analysis of multiple sampling of homogeneous sample. It was demonstrated by repeatability and intermediate precision measurements of peak area and peak symmetry parameters of HPLC method for the title ingredient. The repeatability (within-day in triplicates) and intermediate precision (for 3 days) were carried out at six concentration levels for compound. Triplicate injections were made and the obtained results within and between the days of trials were in acceptable range. The precision expressed as % RSD is given .

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3.4.3.1. Repeatability Six sample solutions were prepared and injected into the HPLC system as per test procedure. Table 3.5. Results of repeatability

Conc. of dasatinib Peak Area (g/mL) 309400 312247 305016 40 307219 307467 308121 0.78 % RSD*

*average

of 6 replicate injections for each concentration

Acceptance criteria Relative standard deviation of percentage assay results should not be more than 2.0 %. Observation The Relative standard deviation was found to be 0.78% .

83

3.4.3.2. Intermediate precession (analyst to analyst variability) Two analysts as per test method conducted the study. For Analyst-1 refer precision (Repeatability) results and the results for Analyst-2 were discussed below. Table 3.6. Results of intermediate precession Conc. of dasatinib (g/mL) 308344 307467 307219 50 305017 312247 309402
*-average RSD of 6 replicate injections

Peak Area % RSD*

0.80

Acceptance criteria Relative standard deviation of % assay results should not more than 2.0 % by both the analysts. Observation The Relative standard deviation was found to be 0.80% . 3.4.4. Accuracy Accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of an analytical method should be established across its linearity range. Accuracy was performed in three different levels, each level in triplicate for

84

Capecitabine using standards at 50%, 100% and 150%.Each sample was analysed in triplicate for each level.

Table 3.7. Percent recovery results for Dasatinib Sample Concentration Amount Amount % of spiked of drug of drug Percent Statistical analysis level found added recovery of %recovery in mg in mg 50 50 50 100 100 100 150 150 150 150 100 50 50.20 50.15 49.90 100.50 100.10 100.20 150.0 149.85 150.09 100.4 100.3 99.8 100.5 100.1 100.2 100.0 99.7 100.19 Mean- 100.16 S.D- 0.109 %RSD- 0.108 Mean- 100.26 S.D- 0.152 %RSD- 0.151 Mean- 99.96 S.D- 0.247 %RSD- 0.247

1. 2. 3. 1. 2. 3. 1. 2. 3.

Acceptance criteria The mean % recovery of the Dasatinib monohydrate at each spike level should be not less than 98.0 % and not more than 102.0 %. Observation: The mean % recovery levels were found to be 100.1.
85

3.4.5. Specificity Specificity is the ability to asses unequivocally the analyte in the presence of components which may be expected to be present.Lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedures. Solutions of standard and Sample are prepared as per test method and injected into the chromatographic system. Blank interference: A study to establish the interference of blank was conducted. Mobile phase was injected as per the test method. Chromatogram of blank should not show any peak at the retention time of analyte peak.

Fig 6 Standard chromatogram for Dasatinib identification

86

Fig 7 Chromatogram for blank interference

Fig 8Chromatogram for placebo interference

87

3.4.6. Robustness The robustness of the proposed method was determined by analysis of aliquots from homogenous lots by differing physical parameters like flow rate and mobile phase composition which may differ but the responses were still within the specified limits of the assay.

3.4.6.1. Effect of variation of flow rate A study was conducted to determine the effect of variation in flow rate. Standard solution was prepared and injected into the HPLC system by keeping flow rates 1.0 ml/min and 1.2 ml/min. The effect of variation of flow rate was evaluated. 3.4.6.2. Effect of variation of temperature A study was conducted to determine the effect of variation in temperature. Standard solution prepared as per the test method was injected into the HPLC system at 25, 27 and 35C temperature. The system suitability parameters were evaluated and found to be within the limits for a temperature changes.

88

Table 3.8.Results of robustness Optimum Parameters range procedure At lower flow rates the asymmetry factor was Flow ml/min retentions was decreased. Beyond the optimum range there is a change Temperature 25,35 C
o

Conditions

in Remarks

rate 1.0,1.2 1.2 increased and at higher flow rates the relative

Ambient in symmetry.

3.4.7. Limit of detection (LOD) Calibration curve was repeated for 5 times and the standard deviation (SD) of the intercepts was calculated. The LOD was determined by the formula: LOD = 3.3 / S = 3.3 (98.1936 / 7985) = 0.0405 Detection limit was 0.0405 g / ml.

89

3.4.8. Limit of quantification (LOQ) Calibration curve was repeated for 5 times and the standard deviation (SD) of the intercepts was calculated The LOQ was determined by the formula: LOQ = 10 / S = 10 (98.1936 / 7985) = 0.1229 Quantification limit was 0.1229 g / ml.

90

RESULTS & DISCUSSION

The objective of the proposed work was to develop a method for the determination of Dasatinib monohydrate to validate the methods according to USP and ICH guidelines and the methods developed was found to be rapid, simple, precise, accurate and economic and then applied on pharmaceutical dosage form. In the method development, HPLC conditions were optimized to obtain, an adequate separation of eluted compound. Various ratios of mobile phase systems were prepared and used to provide an appropriate of of buffer (pH-6.50.05):solvent mixture [acetonitrile: methanol(50:50 v/v)] in the ratio of 50:50v /v is used gave a better resolution and sensitivity. Mobile phase and flow rate selection was based on peak parameters (height, tailing, theoretical plates, capacity or symmetry factor), run time. The optimum wavelength for detection was 315 nm at which better detector response for the title drug was obtained. The retention time for Dasatinib monohydrate was found to be 6.467 min . The calibration was linear in concentration range of 20-60 g mL-1 with regression 0.9999, intercept -11196 and slope 7985 for Dasatinib monohydrate . The low values of % R.S.D indicate the method is precise and accurate. Sample to sample precision and accuracy were evaluated using t samples of different concentrations, which were prepared and analyzed on same day. These results show the accuracy and repeatibility of the assay. The % R.S.D. reported was found to be less than 2 %.The proposed method was validated in accordance with ICH parameters and the applied for analysis of the same in laboratory prepared mixtures.

91

The Limit of Quantification and Limit of Detection were calculated from the linearity curve method using slope and standard deviation of intercepts of calibration curve. Limit of Quantification and Limit of Detection were found to be 0.1229 g / ml and 0.0405 g / ml respectively. The proposed methods are accurate, simple, rapid and selective for the estimation of Dasatinib monohydrate in laboratory prepared mixtures. Hence, these methods can be conveniently adopted for the routine analysis of Dasatinib monohydrate in quality control laboratories.

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SUMMARY & CONCLUSION

A HPLC method was developed for the estimation of Dasatinib in tablet dosage form using reverse phase high performance liquid chromatography.HPLC Waters (Model.No:2690) with UV\VIS detector and Cosmicsil BDS C- 18 with ambient temperature, injection volume of 10l is injected and eluted with mobile phase of phosphate buffer (pH-6.5), Acetonitrile and methanol in the ratio 50:50 v/v, which was pumped with a flow rate 1.0ml/min and detected by UV at 315nm. The peak of Dasatinib was found at 6.4675min.The developed method was validated for various parameters as per ICH guidelines like accuracy, precision, linearity, LOD, LOQ, ruggedness and robustness.The proposed method was applied for the determination of Dasatinib in tablets. Hence the proposed method was found to be satisfactory and could be used for the routine analysis of Dasatinib in the tablets.

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