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

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 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 material’s 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.

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    A standard method for analysis of concentration involves the creation of a calibration curve. precision and speed) and cost (purchase.1.2.1. Several examples were in popular use today and new hybrid techniques were under development. selectivity. If the concentration of elements or compound in a sample is too high for the detection range of a technique. accuracy. 1. Genetic Finger printing and DNA microarray are very popular tools and research filed. it can simply be diluted in a pure solvent. at various development stages or in various parts of the body. Trends Analytical chemistry research was largely driven by performance (Sensitivity. robustness. Methods Analytical methods rely on scrupulous attention to cleanliness.5.Effort was also put into analyzing biological system. accuracy and precision. but dealing with metabolites. operation. 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. 4 .1. 1. linear range.1. training.5.1. Examples of rapidly expanding fields in this area were Genomics DNA sequencing and its related research. sample preparation. Metabolomics Similar to proteomics. Proteomics The analysis of protein concentrations and modifications especially in response to carious stressors. If the amount in sample was below an instruments grange of measurement. time and space).

Peptidomics Peptides and its associated field. 5 . Lipidomics Lipids 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.Transcriptomics mRNA and its associated field.

Choose detector and detector settings settings. 4. Recover purified material 7b. and it should allow the use of sophisticated tools such as computer modeling. sample pre-treatment.6] Every day many chromatographers face the need to develop a High Perfor-mance Liquid Chromatography (HPLC) separation. 3. Finally method development should be as simple.1. Quantitative calibration 7c. as possible. A good method development strategy should require only as many experimental runs as are necessary to achieve the desired final result. Optimize separation conditions 6. sample pretreatment edure. preliminary run.2. Validate method for release to routine laboratory . Need for special HPLC Proceedure. define separation goals 2. Method development often follows the series of steps summarized below: 1. Qualitative method 6 8. ANALYTICAL METHOD DEVELOPMENT[5. Choose LC method. estimate best separation conditions 5. Information on sample. Check for problems or requirement for special procedure 7a.

1.1. 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. 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. Separation goals The goals of HPLC separation need to be specified clearly. Nature of the sample: Before beginning method development.2. The chemical composition of the sample can provide valuable clues for the best choice of initial conditions for the HPLC separation. WHAT IS KNOWN BEFORE STARTING A METHOD DEVELOPMENT A. B. 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. 7 .

The samples may be of two types.  Knowledge on the desired HPLC equipment.2. When the number of samples for analysis at one times is greater than 10. 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. The regular samples are typical mixture of small molecules (< 2000 Da) that can be separated by normal starting conditions.2. since this minimizes base line upset and other problems. Some samples require a partial separation (pretreatment) prior to HPLC. buffering. regular or special. SAMPLE PRETREATMENT AND DETECTION Samples come in various forms:  Solutions ready for injections  Solutions that require dilution. Where as special samples are better separated under customized conditions given in the following table. Best results are often obtained when the composition of the sample solvent is close to that of the mobile phase. HPLC experience and academic training do the operators have? 1. because it is necessary to remove interferences. a run time of less than 20 minutes often will be important. 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. 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. 8 .

Type of samples and their requirements Type of Sample Requirements Inorganic ions Detection is primary problem. DEVELOPING THE SEPARATION A.3. 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.2. (>> 10-nm diameters). Enantiomers Biological compounds These compounds require chiral conditions for their separation. the next step is to classify the sample. as regular or special.Table 1. Several factors make samples of this kind “special” mole molecular conformation.1. Macromolecules “Big” molecules require column packings with large pores. Regular samples are typical mixtures of small molecules that can be separated using 9 . Selecting an HPLC Method and Initial Conditions: If the HPLC is chosen for the separation. and a wide range of hydrophobicity. biological molecule require special conditions 1. in addition. polar functionally.

size of the ions generated by the sample molecules and the nature of ionisable group on the stationary phase. the separation mechanism is based on inductive forces. In case of ionexchange chromatography. 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. 10 . in the adsorption and partition modes. In the case of size-exclusion chromatography the selection of the column is based on the molecular weight and size of the sample components. Special samples are usually better separated with a different column and customized conditions. Selection of columns based on the method is briefly summarized in the following table. dipole-dipole interactions and hydrogen bond formation.more or less standardized starting conditions. the separation is based on the differences in charge.

and an Ion-pair reagent. Ion pair HjPLC Uses water-organic mobile phase. A buffer to control pH. 11 . cyano. first choice for lipophilic samples that do not dissolve well in water-organic mixtures. silica. trimethylsilys (TMS). diol. C8.Table 1. Different methods of HPLC Method /Description /Columns Reversed–phase HPLC Uses water-organic mobile First choice for most samples. C8. Acceptable choice for ionic or ionisable compounds. amino. especially Preferred Method phase Columns: C18 (ODS). dissolve in water-organic mixtures cyano. Columns: C18. 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.2. especially bases or cations. neutral or non-ioniged compounds that Phenyl.

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

bases. Experimental conditions for the initial separation of regular sample Separation Variable Column: Dimensions (length. ID) Stationary phase Particle size 15 x 0.5-2 ml/min 35-45o C < 25 µl < 100 µg 13 . Samples classified as ionic include acids.Regular samples can be further classified as neutral or ionic. Table 1. and organic salts (ionized strong acids or bases).46 cm C8 or C18 5 µm 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 1.3. amphoteric compounds.

For basic or cationic samples. isocratic or gradient elution can be selected as most suitable. Acids or bases usually require the addition of a buffer to the mobile phase. but few groups will separate. At this point it may also be apparent that typical reversed phase conditions provide insufficient sample retention. the sample may be strongly retained with 100% ACN as mobile phase. A better alternative is to use a very strong mobile phase first (80-100%) then reduce % B as necessary. Getting Started on Method Development: One approach is to use an isocratic mobile phase of some average organic solvent strength (50%).If the sample is neutral. suggesting the use of either ion–pair or normal phase HPLC alternatively. the first exploratory run is carried out and then improved systematically. and amine additives for the mobile phase may be beneficial using these conditions. The initial separation with 100% B results in rapid elution of the entire sample. On the basis of the initial exploratory run. 14 . suggesting the use of non aqueous reversed phase chromatography or normal phase HPLC. B. Decreasing the solvent strength shows the rapid separation of all components with a much longer run time. less acidic reversed phase columns are recommended. buffers or additives are not required in the mobile phase. with a broadening of latter bands and reduced retention sensitivity.

Separation or resolution is a primary requirement in quantitative HPLC analysis. 15 .Table 1. baseline resolution (RS >1. Some HPLC assays do not require base line separation of the compounds of interest.5) can be obtained easily for the bands of interest. Separation time Quantitation < 5-10 min is desirable for routine procedures.4. < 200 bar is usually essential (new column assumed) Peak height Solvent consumption Narrow peaks are desirable for large signal/noise ratios. it may be tempting to accept a marginal separation. Minimum mobile-phase use per run is desirable. values of RS = 2 or greater should be the goal during method development for simple mixtures. < 15% for trace analysis Pressure < 150 bar is desirable. After a few additional tries. for samples containing five or fewer components. Resolution usually degrades during the life of the column and can vary from day to day with minor fluctuations in separation conditions. In such cases only enough separation of individual components is required to provide characteristic retention times for peak identification. The separation achieved in the first one or two runs usually will be less than adequate. Such resolution will favor both improved assay precision and greater method ruggedness. Therefore.5. This level of resolution favors maximum precision in reported results. < 5 % for less-demanding analysis. < 2% (ISD) for assays.Goals that are to be achieved in method development Goal Resolution Comment Precise and rugged quantitative analysis requires that RS be greater than 1. especially if no further improvement is observed. Usually.

However. column equilibrium is achieved after passage of 10-20 column volumes of the new mobile phase through the column. columns tend to plug less. When changing conditions (mobile phase. 17 MPa). despite the fact that most HPLC equipment can be operated at much higher pressures. enough time must elapse for the column to come into equilibrium with the new mobile phase and temperature. due to the gradual plugging of the column by particulate matter. column. Second. a run time of 20-30 min is not excessive. and system reliability is significantly improved. during the life of a column. There are two reasons for this pressure limit. The run time goal should be compared with the 2-h setup time typically required for an HPLC procedure. For these reasons. This assumes that the other goals of previous table have been achieved. this should be confirmed by carrying out a repeat experiment under the same conditions. sample valves. 16 . experiments. the back pressure may rise by a factor of as much as 2. longer equilibration times can result when one of the two mobile phases being interchanged contains <10% organic. C. seals last longer. it is important to confirm that each chromatogram can be repeated. 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.The time required for a separation should be as short as possible. When dealing with more challenging samples or if the goals of separation stringent. 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. run times of 5 to 10 min are desirable. and especially auto samplers operate much better. and an upper pressure limit below 2000 psi desirable. Thus if only two or three samples are to be assayed at one time. For reversed-phase separations. pumps. temperature) between method developments. a large number of method developments run may be required to achieve acceptable separation. When lots of 10 or more samples are to be assayed. Repeatable Separation As the experimental runs described above are being carried out. a target reassure of less than 50% of the maximum capability of the pump is desirable. First. Usually. and the total time spent on method development is reasonable. at lower pressures (< 170 bar).

3. Deviant results studied for possible correction of hidden problems.1. the method should also robust in routine operation and usable by all laboratories and personnel for which it is intended Completing the Method 1.) studied for effect on separation. limits defined for these variables. etc.2. 4. COMPLETING THE METHOD DEVELOPMENT The final procedure should meet all the goals of the method development. increased retention for last band with longer run times. using samples that cover the expected range in composition and analyte concentration.). All variables (temperature. 5. mobile phase composition. Data obtained on expected life of column and column-to-column reproducibility. Preliminary data to show required method performance. 6. data obtained for day to day and inter laboratory operation. 17 . etc. remedies suggested for possible problems (poor resolution of key band pair. Written assay procedure developed for use by other operators.4. 2. Systematic validation of method performance for more than one system or operator.

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.1 High Performance Liquid Chromatography High Performance Liquid Chromatography (HPLC) is one mode of chromato -graphy. In the column.3. are resolved by sorption-desorption steps on the stationary phase. As a result. the most widely used analytical technique HPLC utilizes a liquid mobile phase to separate the components of a mixture. 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. 1. INTRODUCTION TO HPLC [7-10] Chromatography: Chromatography is a technique by which the components in a sample. carried by the liquid or gaseous phase.1. reusable stainless steel columns Column packing with very small (3. These components (or analytes) are first dissolved in a solvent. and then forced to flow through a chromatographic column under a high pressure. 5 and 10 µm) 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 18 .1. HPLC as compared with the classical technique is characterized by      Small diameter(2-5 mm).

the longer analyte will be retained on the surface. Basic principle of SEC separation is that the bigger the molecule. while moving through the porous packing bead. tend to interact with the surface adsorption sites. Analyte molecules compete with the molecule at adsorption sites. Analyte molecules. 19 . the less possibility for her to penetrate into the adsorbent pore space. In this mode any positive surface interactions should be avoided. HPLC is a dynamic adsorption process. It is the separation of the mixture by the molecular size of its components. So the stronger analyte molecules interacts with the surface and the weaker the eluent interaction. SEC (size-exclusion chromatography) is a special case. All these interactions are competitive. 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.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.   Dipole-dipole (polar) interactions are dominated in normal phase mode Ionic interactions are responsible for the retention in ion-exchange chromatography. so. Depending on the HPLC mode. the bigger the molecule the less it will be retained.

Based on modes of chromatography: 1. it is non-polar or the nature of the phase is reversed. Reverse Phase Chromatography The stationary bed is non polar (hydrophobic) in nature.1. Based on principle of separation:      C. Here the more non polar the material is. Based on the scale of operation:   Analytical HPLC Preparative HPLC A.. Since the ionic nature of the chemically modified silica in now reversed i.1. the chromatographic separation carried out with such silica is referred to as reversed phase chromatography. 20 . the longer it will be retained. such as mixtures of water and methanol or acetonitrile. TYPES OF HPLC TECHNIQUES A.2.e. The object was to make silica less polar or non-polar so that polar solvents can be used to separate water-soluble polar compounds. Based on modes of chromatography:   Reverse phase chromatography Normal phase chromatography B. Based on elution technique:   Isocratic separation Gradient separation Adsorption chromatography Ion exchange chromatography Size exclusion chromatography Affinity chromatography Chiral phase chromatography D. while the mobile phase is a polar liquid.

Since most pharmaceutical compounds are polar and water soluble. The retention time decreases in the following order: Aliphatic > induced dipoles (E.A large number of chemically bonded silica based stationary phases are available commercially. Branched chain compounds are eluted more rapidly than their corresponding normal isomers. As general rule the retention increases with an increase in the contact area between sample molecule and stationary phase i. which are released during the adsorption of a compound. decomposition studies. 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. Only higher polar or ionic solutes can interact with the water structure. adsorption of sample molecules become highly restricted and they are rapidly eluted as a result. CHCl3) > weak Lewis bases (Ethers. Water interacts strongly and highly with silanol groups. the majority of HPLC methods used for quality assurance. 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. phenols) > strong Lewis acids (carboxylic acids ).g. The solvent strength in reverse phase chromatography is reversed from that of adsorption chromatography (silica gel) as stated earlier. The less water–soluble compounds are better retained by the reversed phase surface. aldehydes. water cannot wet the non-polar (hydrophobic) alkyl group such as C18 of ODS phase and therefore does not interact with the bonded moiety. Silica based stationary phases are still more popular in reversed phase chromatography. Chemically bonded octadecyl silane (ODS) and alkane with 18 carbon atoms is the most popular stationary phase used in pharmaceutical industry. Hence water is the weakest solvent of all and gives slowest 21 . Exactly opposite applies in reversed phase system.e. Also the retention increases as the number of carbon atoms increases.. CCl4) > permanent dipoles (E. with an increase in the number of water molecules. quantitative analysis of both bulk drugs and their formulations use ODS HPLC columns. so that.g. however other adsorbents based on polymer (styrene divinyl benzene copolymer) are slowly gaining ground. ketones) > strong Lewis bases (amines ) > weak Lewis acids (alcohols.

Size Exclusion Chromatography The column is filled with material having precisely controlled pore sizes. The stronger the charge on the sample. 22 . where adsorption takes place. and the sample is simply screened or filtered according its solvated molecules. Separation of components takes place because of the difference in affinity of compounds towards stationary phase. B. Adsorption Chromatography The principle of separation is adsorption.elution rare. The elution time (retention time) in reversed phase chromatography increases with increasing amount of water in the mobile phase. This technique is used almost exclusively with ionic or ionizable samples. The mobile phase is an aqueous buffer. the stronger it will be attracted to the ionic surface and thus. where both pH and ionic strength are used to control elution time. 3. Large molecules are rapidly washed through the column smaller molecules penetrate inside the porous of the packing particles and elute later. 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. Based on principle of separation: 1. Ion Exchange Chromatography The stationary bed has an ionically charged surface of opposite charge to the sample ions. 2. 2. This principle is seen in normal phase as well as reverse phase mode. the longer it will take to elute.

Based on elution technique: 1. 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. Affinity chromato-graphy. 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. C. Isocratic Separation In this technique the constant eluent composition is pumped through the column during the whole analysis. common for macromolecules. Recovery of the samples for reusing is normally not done. Affinity / Ion. employs a ligand (biologically active molecule bonded covalently to the solid matrix).4.Pair Chromatography Separation is based on a chemical interaction specific to the target species. the sensitivity is enhanced if the enantiomeric impurity elutes before the enantiomeric drug. Gradient Separation In this technique the eluent composition (and strength) is steadily changed during the whole analysis. Based on the scale of operation: 1. 23 . concentration and type of organic co-solvent(s). temperature. 5.Which interacts with its homologous antigen (analyte) as a reversible complex that can be eluted by changing buffer conditions. 2. Analytical HPLC In this only analysis of the samples are done. ionic strength. since the samples used are very low. D. When used as an impurity test method.

Preparative HPLC Where analysis of the individual fractions of pure compounds can be collected using fraction collector.1. INSTRUMENTATION Fig-1: HPLC Instrument In order to realize eluent flow rates with packing in the 2 to 10 µm particle sizes.2.3. 1. which are common in modern liquid chromatography. As a consequence of these high pressures. The collected samples are reused. pumping pressures of up to several thousand pounds per square inch are required. the equipment 24 .

required for HPLC tends to be more elaborate and expensive than that encountered in other types of chromatography. Size-exclusion HPLC has special requirements. the adsorbent could be normal phase (-OH-NH2). B. and even anion (NH4+). C18. Stationary Phases (Adsorbents) HPLC separations are based on the surface interactions. Despite of the large variety of solvents used in HPLC. or reversed-phase (C8. Modern HPLC adsorbents are the small rigid porous particles with high surface area Main adsorbent parameters are:    Particle size: 3 to 10 µm Particle size distribution: As narrow as possible. 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 reversed-phase eluents are usually a mixture of water with some polar organic solvent such as acetonitrile. or cation (-COO) exchangers. For normal phase mode solvents are mainly non polar. and depend on the types of the adsorption sites (surface chemistry). usually within 10% of the mean Pore size: 70 to 300 Å Depending on the type of the ligand attached to the surface. Mobile phase (eluents) In HPLC type and composition of the mobile phase (eluent) is one of the variables influencing the separation. A. Phenyl). SEC eluents 25 .

Helium purging and storage of the solvent under helium was found not to be sufficient for degassing of aqueous solvents. column. Mobile phase reservoir. It is desirable to have an integrated degassing system. Flow rate stability is another important pump feature that distinguishes pumps. for size exclusion chromatography the flow rate has to be extremely stable. 26 . 340 atm). The constant-flow pump is the most widely used in all common HPLC applications. High pressure pumps are needed to force solvents through packed stationary phase beds. For most types of separation stable flow rate is not very important.01 to 10 ml/min Flow rate stability: Not more than 1% (short term) For SEC flow rate stability should be less than 0. The constant-pressure pump is used only for column packing. The two basic classifications are the constant-pressure and the constant-flow pump. or better vacuum degassing. D. many separation problems can be resolved with larger particle packing that requires less pressure. 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. 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. either helium purging. C. Teflon tubing and filters to connect to the pump inlet and to the purge gas (helium) used to remove dissolved air. However. Modern pumps have the following parameters:     Flow rate range: 0. Most of the manufacturers supply these bottles with the special caps. and then keep the solvent under a helium atmosphere.has to dissolve polymers.2% Maximum pressure: Up to 5000 psi (345 bar. and detector. filtering The most common type of solvent reservoir is a glass bottle. However. It is useful to apply a vacuum for 5-10 min.

and loss in efficiency or all of these. this is considered the best compromise among sample capacity. Packing of the column tubing with the small diameter particles requires high skill and specialized equipment. In liquid chromatography. automatic sampling devices are incorporated where sample introduction is done with the help of auto samplers and microprocessors.They should also produce minimum band broadening and minimize possible flow disturbances. The internal diameter of the columns is usually 4 or 4. The solvent need not be the mobile phase. liquid samples may be injected directly and solid samples need only be dissolved in an appropriate solvent. In more sophisticated LC systems. The most useful and widely used sampling device for modern LC is the micro sampling injector valve. The simplest method is to use an injection valve. column/component interference. F. speed and resolution.1 to100 ml of volume with high reproducibility and under high pressure (up to the 400 psi). For this reason. Columns The heart of the system is the column. 5 or 10 µm) particles.E. Typical analytical columns are 10. but frequently it is judiciously chosen to avoid detector interference. since it is difficult to match the high performance of professionally packed LC columns without a large investment in time and equipment. 27 . it is generally recommended that all but the most experienced chromatographers purchase pre-packed columns. It is always best to remove particles from the sample by filtering.6 mm. Preparative columns are of larger diameter. Injectors should provide the possibility of injecting the liquid sample within the range of 0. or centrifuging since continuous injections of particulate material will eventually cause blockage of injection devices or columns. mobile phase consumption. 15 and 25 cm in length and are fitted with extremely small diameter (3. Injectors Sample introduction can be accomplished in various ways.

however. The two chromatograms may be compared to establish whether or not the column is still used. the particle size is usually larger. with highly acidic or basic eluents. 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. it is repacked or discarded and replaced with a new one of the same type. G. the guard column is a sacrificed to protect the more expensive analytical column. however. 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. Columns may also be fitted with water jackets fed from a constant temperature bath to give precise temperature control. In addition. Thus. and to retain the chromatogram.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. and columns are operated at ambient temperature. Often. as for example. For many applications. When the guard column has become contaminated. 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.grams are obtained by maintaining column temperatures constant to a few tenths degree centigrade. to minimize pressure drop. If questionable results are obtained later the test mixture can be injected again under specified conditions. in liquidliquid chromatography. It is wise to inject some test mixture (under fixed conditions) into a column when new. or with continual injections of 'dirty' biological or crude samples. close control of column temperature is not necessary. better chromato. Detectors The function of the detector in HPLC is to monitor the mobile phase as it emerges from the column. 28 .

Choosing a Detector: Table 1. 2. Types of Detectors RI Response Sensitivity Flow sensitive Temp. flow rate. It is also dependent on the standard deviation of the measurements. 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. 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. Detector sensitivity Detector sensitivity is one of the most important properties of a LC detector. 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. but high fluctuations of your measurements will decrease the sensitivity. Sensitivity can be associated with the slope of the calibration curve.1. 29 .5. The higher the slope of your calibration curve the higher the sensitivity of your detector for that particular component.

bulk property detectors and solute property detectors. Types of Detectors Generally. and dielectric constant detectors. there are two types of HPLC detectors. such as refractive index. fluorescence.  Bulk property detectors: These detectors are responding to a mobile phase bulk property. the response R (mV/mass/unit volume) is: Where: h = peak height (mV) W = peak width at 0.4. Optical detectors are most frequently used. Response For mass-sensitive detectors. Such as UV absorbance. These detectors pass a beam of light through the flowing column effluent as it passes through a low volume (~ 10 ml) flow cell. the response R (mV/mass/unit time) is: R = hW / sM For the concentration sensitive detector. 30 . which is not exhibited by the pure mobile phase.  Solute property detectors: Solute property detectors respond to some property of the solutes.607 of the peak height (cm) F = M = s = flow rate (ml/min) mass of solute injected chart speed (cm/min) 5.

Analytes must have fluorophore group. The detector usually contains low volume cell through which the mobile phase passes carrying the sample components.The most commonly used detector in LC is the ultraviolet absorption detector. while reducing operator attention. H. Several types of ionization techniques: electrospray. Sometimes difficult to stabi-lize baseline. 31 .Excitation wavelength generates fluorescence emission at a higher wavelength. Allows specific compound ID. a pre-programmed computing integrator may be sufficient.Mass to charge ratio (m/z). Results very dependent upon separation conditions. Can react analyte with fluorophore reagent.  Other types of detectors RI – Refractive Index-Universal analyte detector. Very temperature sensitive.4. Very sensitive and selective. atmospheric pressure chemical ionization. where no automation (in terms of data management or process control) is needed. electron impact.8] A. capable of monitoring from 190 to 460-600nm.1. In routine analysis. FD – Fluorescence. 1. PARAMETERS USED IN HPLC [ 5. Solvent must remain the same throughout separation. More difficult methods transfer. MS – Mass Spec. will be found suitable for the detection of the majority samples. Data systems The main goal in using electronic data systems is to increase analysis accuracy and precision. A variable wavelength detector of this type. 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.

ta) / (t1 . w1 and w2 are their baseline band widths. Retention time of the first peak measured from point of injection. α t2 t1 ta = = = = Relative retention Retention time of the second peak measured from point of injection.5. C. W0.t1) / (W0. Retention time of an inert peak not retained by the column measured from point of injection B.1 and W0.1 + W0. It is the product of retention time and flow rate.2) 32 .t1) / (w1 + w2) Where.ta) Where.5.5.5.α = (t2 . Rs = 2(t2 .18(t2 . t1 and t2 are the retention times of the first and second adjacent bands.2 then calculations of Rs using above equation (or) below equation may not be reliable when Rs is less than 1. Retention volume Retention volume is the volume of mobile phase required to elute 50% of the component from the column. An alternative approach gives more reliable values of Rs band widths at half height (w1/2) are measured for bands 1 and 2. Resolution (Rs) Resolution is measure of the extent of separation of two components and the base line separation achieved. Rs = 1.

1) N1/2 K / (1 + k) Where. Rs = 2(t2 .t1) / (w1 + w2) 2. column (N) and selectivity (α).Resolution can be estimated or measured in 3 different ways: 1. K N α α = = = = 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. Rs = 1/4(α . The above equation is useful in method development because it classifies the dozen (or) so many experimental variables into 3 categories: retention (k). 3. 33 . α. Calculations based on below e.q. Resolution can be expressed in terms of three parameters (k. Calculations based on the valley between the 2 bands. and N) which are directly related to experimental conditions. Comparison with standard resolution curves.

A theoretical plate is an imaginary or hypothetical unit of a column where equilibrium has been established between stationary phase and mobile phase. which refers to the mass of the solute that a specified amount of medium is capable of binding under defined conditions. 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. using the following equations. Long retention times results in large values of K. Column dead time or Column void time solvent peak E. K1 = tR . N is dimensionless number and reflects the kinetics of the chromatographic retention mechanism. The capacity factor is not the same as the available binding capacity. 1.t0/t0 Where. 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. Column efficiency Two related terms are widely used as quantitative measures of chromatographic column efficiency. 34 . tR t0 = = Retention time of a solute peak. The capacity factor K1can be calculated for every peak defined in a chromatogram. Plate height 2. Efficiency depends primarily on the physical properties of the chromatographic medium together with the chromatographic column and system dimensions.D.

5. Column performance can be defined in terms of values of N and band asymmetry (band shape) for a test substance run under “favorable” conditions. 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. 3.54(tR / W1/2)2 Here. 2. The column plate number increases with several factors: 1.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. tR W1/2 = = is band retention time is the band width at half height. 7. 35 . A theoretical plate can also be called as a functional unit of the column. Therefore a more practical equation for N is N = 5. 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. 4. 6.

4. they readily validate. 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 .    When methods are properly developed. The following observation will explain the relationship between validation and method development. 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].1. 36 .S. The U. Validation acceptance criteria should be based on method development experience. Validation is not a method development tool and it does not make a method good or efficient.

Intermediate Precision and Reproducibility. 1. Precision: The closeness of agreement between a series of measurements multiple samplings of the same homogeneous sample under prescribed condition. 37 .1. The precision of test method is usually expressed as the standard deviation or relative standard deviation of a series of measurements. 4. Precision may be considered at three levels: Repeatability.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.

Accuracy has also been reported as a sample is analyzed and the measured value should ideally be identical to the true value. 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. a – Amount of drug present in sample b – Amount of standard added to the sample. That is the accuracy or % recovered is calculated as: Cm × 100 Ct Where Cm is the measured concentration and Ct is the theoretical concentration. The % recovery was calculated using the formula. % Re covery  ( a  b)  a bX 100 Where. 38 . In this case.2.4. There are several methods that can be used for determining accuracy. The usual range is being 10% above or below the expected range of claim.Acceptance Criteria:   Percentage Relative standard deviation (%RSD) NMT 1 % (Instrument precision) (%RSD) NMT -2% (Intra. 1996). method accuracy is the agreement between the difference in the measured analyte concentration and the known amount of analyte added. 2003). 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. The most common include: Analyze a sample of known concentration and compare the measurement to the true value. Accuracy is represented and determined by recovery experiments.assay precision) 1.

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 (σ) 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(σ) 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

Neutralize the solution and dissolve the contents in diluents as per test method. Dissolve the contents in diluents as per test method.  Water: Reflux the sample / placebo with 100 ml of purified water for 12 hour at 60°C.1N and reflux the sample and placebo with 50 ml of acid / base solution for about 1 hour at 60°C. Peak purity of analyte peak should be confirmed.  Humidity: Expose the Drug product. Degradation of active analyte peak should be from 1% to 20%. 42 .  Heat: Expose the Drug product.  Oxidation: Reflux for 12 hour at 60°C with 1 % H2O2 or suitable oxidant. Light: Expose the Drug product. Note: Based on the physicochemical properties and literature stress conditions can be decided. drug substance and placebo for about 80 % RH at about 25°C for about one week. Change the strength of acid and base or reflux time to ensure the desired degradation.  Acid / Base: Prepare the acid or base solution of 0. Change the reflux time so as to ensure the desired degradation. Dissolve the contents in diluents as per test method.2 million Lu hours [17] for visible light. Prepare the sample and placebo solution as per test method and analyse. Change the reflux time so as to ensure the desired degradation. Prepare the sample and placebo solution as per test method and analyse. drug substance and placebo at 105 °C for about 12 hours ( For substance having low melting point below 10°C of its melting point ). Prepare the sample and placebo solution as per test method and analyse. Acceptance Criteria:    Placebo / Blank should not elute at the retention time of analyte peak and known impurity peak. drug substance and placebo to UV light for about 200 watt hours / square meter and the overall illumination not less than 1.

4. 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.7.0. (+/. X is the concentration of sample. 1. For the method to be linear the r value should be close to1.1.99. establish the range of concentration in which it is linear. a is the slop. 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 : : : : : (+/.10% of organic phase). Acceptance criteria: Coefficient of correlation should be NLT 0. 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. 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. 43 . If linearity is not meeting the acceptance criteria.6. (At least two filters). (+/. Where Y is the measured output signal. 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. 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.2ml/minutes).2 units).4. b is the intercept. The linear range of detectability that obeys Beer’s law is dependent on the compound analyzed and the detector used.5°C). (+/. For example a chromatographic method. The working sample concentration and samples tested for accuracy should be in the linear range.0. accuracy and linearity using the method as written.

Different Laboratories.4. Stability of Solutions and mobile phase. 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. Column-to-Column variability. Ruggedness is a measure of Reproducibility of test results under normal. 2. different analysts. ( At least for 48 hours ) 44 .For Variations: 1. If system suitability doesn’t meet. expected operational conditions from laboratory to laboratory and from analyst to analyst”. narrow the variation range and carryout the experiment again to meet system suitability.8. 1. Different days. such as different labs. and different lots of reagents. The following are the typical method parameters need to be tested during method validation:       Analyst-to-Analyst variability. System suitability should meet the acceptance criteria as per test method. System-to-System variability.

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 100.99 ± 10% Linear yes ± 2% change from time zero >2 min.6.Table 1.0% ≥ 3:1 ≥ 10:1 >0.0% ± 2. (retention time) 45 .

 The precision or reproducibility of the analytical method was determined by repeating the analysis and the following statistical parameters were calculated. 1.1.1 Mean Best estimation of the population mean mcg/ml for random samples from a population.5.5.5.x 100 Mean 46 . STATISTICAL PARAMETERS Statistics consist of a set of methods and rules for organizing and interpreting observations.2 Standard deviation The positive square roof of the variance S.D = 1. ̅ Where ∑ ̅ x n = = = = Sum of observations Mean or arithmetic average (Ex/n) Individual observed valve Number of observation ∑ 1.3 Relative standard deviation / Coefficient of variation Measures of the spread of data compared with the mean SD  RSD = -----.5.

5. When the concentration range was so wide that the errors.00 Correlation value was denoted with the letter r n(xy) – (x)( y) r=  (nx2 – (x)2 (ny2 – (y)2 1. When paring was inappropriate for other reason. both random and systematic were not independent (which was assumption).6 Regression Purpose : 1.4 Standard error SE = SD / n E n S.D = = = Sum of observations Number of observation Standard deviation 1.) 47 .5. 2.1..5. notably a long time span between two analysis (sample aging. change in laboratory conditions etc.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.00 + 1. Correlation co-efficient can range from -1.

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 .Regression line Y = mx + b Where.

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. 49 . Hence it is proposed to improve the existing methods and to develop new method for the estimation of dasatinib monohydrate in pharmaceutical dosage forms.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. accurate and precise. Objective The scope of developing and validating an analytical method is to ensure a suitable method for a particular analyte should be more specific. but the existing methods are inadequate to meet the requirements. The main objective for that is to improve the conditions and parameters.

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.PLAN OF WORK   To obtain thorough knowledge in practical HPLC method development.   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. 50 .

1.1.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).408 g/cm3 Solubility. It is being evaluated for use in numerous other cancers. including advanced prostate cancer.1. Physical and Chemical Properties:[22]        Colour.28 51 .Off-white to pale yellow Form -powder Odour-none Density ~ 1.8 pH 7.2.5 (n-octanol/water)1. Structure Systematic name N-(2-chloro-6-methylphenyl)-2-({6-[4-(2-hydroxyethyl)piperazin-1-yl]-2methylpyrimidin-4-yl}amino)-1.8 (acidic group(s))10.3-thiazole-5-carboxamide 2.log Pow ~ 4. 2. Dasatinib is an oral multi. 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.1. DRUG PROFILE: Dasatinib[21] is a 2-aminothiazole-derived inhibitor of Src family kinases. Ethanol and Methanol Partition coefficient.1.4 Dissociation constant-pK1 = 8.1.Soluble in Dimethyl Sulfoxide.

LCK. Absorption  Maximum plasma concentrations (Cmax) of dasatinib are observed between 0. c-KIT.1. dasatinib is predicted to bind to multiple conformations of the ABL kinase.      Melting temperature-280-2860c Molecular Formula . Pharmacokinetics: The pharmacokinetics of dasatinib have been evaluated in healthy subjects and in patients with leukemia. at nanomolar concentrations. YES. and PDGFRß. inhibits the following kinases: BCR-ABL. activation of alternate signaling pathways involving the SRC family kinases (LYN. SRC family (SRC.  Dasatinib exhibits dose proportional increases in AUC and linear elimination characteristics over the dose range of 15 mg to 240 mg/day.2.C22H28ClN7O3S Molecular Weight-506. and multi-drug resistance gene overexpression. Dasatinib inhibited the growth of chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) cell lines overexpressing BCR-ABL. PHARMACOLOGY OF DASATINIB[23] Mechanism of action: Dasatinib. Category-Anti-Cancer. dasatinib was able to overcome imatinib resistance resulting from BCR-ABL kinase domain mutations.02082 [g/mol] CAS number. FYN).1. Based on modeling studies. 52 . HCK).5 and 6 hours (Tmax) following oral administration.863127-77-9 Storage -Store solid and solution at -20° C. 2. Under the conditions of the assays.  The overall mean terminal half-life of dasatinib is 3–5 hours. EPHA2.

dasatinib was a weak time-dependent inhibitor of CYP3A4.   In human liver microsomes. Elimination  Elimination is primarily via the feces. dasatinib has an apparent volume of distribution of 2505 L. represents approximately 5% of the dasatinib AUC. respectively. suggesting that the drug is extensively distributed in the extravascular space. with no concentration dependence over the range of 100–500 ng/mL. The exposure of the active metabolite. Metabolism  Dasatinib is extensively metabolized in humans. 53 . within 10 days. approximately 4% and 85% of the administered radioactivity was recovered in the urine and feces.  Binding of dasatinib and its active metabolite to human plasma proteins in vitro was approximately 96% and 93%. respectively.  Unchanged dasatinib accounted for 0.Distribution  In patients. with the remainder of the dose being metabolites. respectively. Following a single oral dose of [14C]-labeled dasatinib. CYP3A4 was the primary enzyme responsible for the formation of the active metabolite. primarily by the cytochrome P450 enzyme 3A4.  Flavin-containing monooxygenase3 (FMO-3) and uridine diphosphate- glucuronosyltransferase (UGT) enzymes are also involved in the formation of dasatinib metabolites.1% and 19% of the administered dose in urine and feces. which is equipotent to dasatinib.

54 . they should be swallowed whole.Drug-Drug Interactions:   Dasatinib is not an inducer of human CYP enzymes. 2A6. abdominal pain and vomiting. 2D6. 2C9. Tablets should not be crushed or cut. itraconazole. nausea. or 2E1. At clinically relevant concentrations. saquinavir. and bleeding events. 2C8.. indinavir. gastrointestinal events including diarrhea. Dosage and Adminstration: The recommended dosage of dasatinib is 140 mg/day administered orally in two divided doses (70 mg twice daily [BID]). telithromycin may decrease metabolism and increase concentrations of dasatinib . nelfinavir. clarithromycin. It is a time-dependent inhibitor of CYP3A4 and may decrease the metabolic clearance of drugs that are primarily metabolized by CYP3A4. Adverse Reactions:  The most frequently reported adverse events included fluid retention events such as pleural effusion.famotidine induce CYP3A4 enzyme and decrease the plasma concentration of dasatinib. dasatinib does not inhibit CYP 1A2. 2B6. Drugs That May Decrease Dasatinib Plasma Concentrations  Drugs like antacids. 2C19. one in the morning and one in the evening with or without a meal. ritonavir. erythromycin. atazanavir. nefazodone. Drugs That May Increase Dasatinib Plasma Concentrations  Drugs that inhibit dasatinib CYP3A4 are ketoconazole.

. swelling. rash..Side Effects: Headache. muscle pain. redness and pain inside the mouth. dizziness .peeling skin. burning or tingling in the hands or the feet.2 ANALYTICAL PROFILE: 55 .joint pain. tiredness. weakness. 2. pain.1. skin redness . mouth sores etc.

LITERATURE REVIEW: 1. The method is precise (inter-day CV%: 1.Sandra Roche et al. pH 4 (54:46.2 SELECTION OF METHOD The selection of method depends upon the nature of the sample. The method was validated according to FDA recommendations. at a flow rate of 0. His study includes Cellular samples were extracted with a tert-butyl methyl ether:acetonitrile (3:1.9 mL/min at 35°C.0 mm 3 μm) column with isocratic elution using a mobile phase of acetonitirile–10 mM ammonium formate. and reliable high-performance liquid chromatography (HPLC)-UV method for quantification of major tyrosine kinase inhibitors. nilotinib. dasatinib. followed by rinsing and re-equilibration to the initial solvent composition up to 20 min. sunitinib.9%) and sensitive (lower limits of quantification comprised between 1 and 10 ng/mL). 2. Haouala et al. imatinib.3%. mean accuracy is 13. in human plasma is presented.1.2 mL/min. dasatinib. Elisa pirro et al. 3 . 2.2. eluate is monitored at 267 nm. [25] in the year 2009 studied Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib. The limit of detection and limit of quantification for lapatinib was determined to be 15 and 31 pg on column. Separation was achieved on a Hyperclone BDS C18 (150 mm × 2. rapid. Mean intra-day and inter-day precision for all compounds are 2. v/v).2.2 and acetonitrile containing 1% formic acid.. His study includes Reverse-phase chromatographic separation of TKIs is obtained using a gradient elution of 20 mM ammonium formate pH 2.3–9.. accurate (−9. [24] studied Development of a high-performance liquid chromatographic–mass spectrometric method for the determination of cellular levels of the tyrosine kinase inhibitors lapatinib and dasatinib. its molecular weight and solubility.5 and 13. v/v) mixture.2 to +9. extraction recovery ranges 56 .4%). sorafenib and lapatinib by LC tandem mass spectrometry. Chromatographic separation of the drugs is achieved on an RP-C18column at flow rate of 0. Literature survey also helps for the selection of suitable method for the analytical method development of dasatinib in its pharmaceutical dosage form. v/v):1 M ammonium formate pH 3. respectively.5 (8:1.[26]studied Development and validation of simple.9%. and nilotinib.

005 µg/mL. at flow rate of 0. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water + formic acid 0. Antonio D’Avolio et al. extraction recovery ranged within 79 and 91%. The assay was validated over a concentration range of 1. Analytes and the stable labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. mean accuracy was −3. Limits of detection are 50 ng/mL for dasatinib.20%. Calibration curves range from 10 to 0.00–1000 ng/mL for dasatinib and its two active metabolites.[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. 100 ng/mL for dasatinib.and inter-assay precision values for replicate QC control samples were within 5.24 and 81. Michael T. Assay recoveries were high (>79%) . A simple protein precipitation extraction procedure was applied on 250 μl of plasma aliquots. 6. The limit of quantification was set at 0.25 mL/min. To enable reliable quantification of dasatinib and its pharmacologically active metabolites in human plasma during clinical testing. Silvia De Francia et al.3% for all analytes during the assay validation. Furlong et al. Mean intraand inter-day precision for all compounds were 8. a sensitive and reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated. 4. dasatiniband nilotinib. Calibration curves ranged from 50.0 to 0. A simple PBMC isolation and extraction procedure were applied on 10–14 mL of blood aliquots. Samples were prepared using solid phase extraction on Oasis HLB 96-well plates. Intra.25 ng for all the analyzed drugs.05%) on a C18 reverse phase analytical column with 25 min of analytical run. Chromatographic separation of drugs and Internal Standard (quinoxaline) 57 . Chromatographic separation was achieved isocratically on a Luna phenyl–hexyl analytical column.0% of nominal values for all analytes.[28] studied Dasatinib (Sprycel®) is a potent antitumor agent prescribed for patients with chronic myeloid leukemia (CML).76 and 12. Mean QC control accuracy values were within ±9.within 40. limits of quantification .[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.86%. 5.25 ng.81 %.

mean accuracy was 1.[31] studied SRC is a tyrosine kinase that plays a role in oncogenic. UK) under isocratic mobile phase conditions of methanol:50 mM ammonium acetate (pH 8) (65:35. i. It has therefore been prioritized as a candidate therapeutic target in patients with solid tumors. including cytotoxic agents and targeted therapies. breast and glioma. After solid phase extraction the drug mix was separated through a Gemini C6-phenyl column (150 mm × 4. John Araujo et al.5 ng/ml. in a range of solid tumors. and inter-day were <4% and <9%.d. Dasatinib has additive or synergistic activity in combination with a number of other agents. Calibration curves ranged from 10. Emerging clinical data with dasatinib support experimental observations. extraction recovery ranged within 95 and 114%. inhibiting the processes of cell proliferation. 58 . Glivec®). 7.5%. Several SRC inhibitors are now in development.4%. including prostate. both as a single agent and as combination therapy. The limit of quantification was set at 62. have shown that that dasatinib acts as a cytostatic agent. For all compounds the intra-day coefficient of variation and bias were <3% and <5% respectively. v/v) with ultra-violet (UV) detection at 260 nm wavelength. Future clinical trials will further assess the clinical value of SRC inhibition with dasatinib..000 to 62. providing a rationale for combination treatment in a clinical setting.. at flow rate of 1 ml/min. from human plasma. 5 μm) (Phenomenex®. Tasigna®) and imatinib (STI571. CGP-74588.3 and 11. Dasatinib also inhibits the activity of osteoclasts. together with its main metabolite. nilotinib (AMN107.05%) on a C18 reverse phase analytical column with 20 min of analytical run. which have a major role in the development of metastatic bone lesions. invasion and metastasis. Preclinical studies in a wide variety of solid tumor cell lines. of which dasatinib has been most explored. 8.5 ng/ml for dasatinib and nilotinib.was achieved with a gradient (acetonitrile and water + formic acid 0. Andrea Davies et al. with preliminary phase 1 and 2 data demonstrating activity. invasive and bone-metastatic processes.6 mm.[30] studied A high performance liquid chromatography (HPLC) method that separates two of the currently licenced tyrosine kinase inhibitors (TKIs). Mean intraday and inter-day precision for all compounds were 4.

wet heat and photo-degradation.d.as well as on inter-day basis.1% formic acid in methanol. accuracy. PerfectSil column [C18 (5 μm.6 mm.[32] studied Two sensitive and reproducible methods are described for the quantitative determination of dasatinib in the presence of its degradation products. i. absolute and relative matrix effect and stability.5 μg/L for dasatinib.3%) on intra.6 min.02).997.)] at ambient temperature using mobile phase consisting of methanol:20 mM ammonium acetate with acetic acid (45:55. The method was validated in terms of linearity. No chromatographic interference from the tablet excipients was found.2) and precise (CV < 10. The drug was found to be stable in neutral. The effect of haematocrit (Hct) on the accurate concentration determination was also examined.0:3.02 min). dry heat. 10. Lower limits of quantification (LLOQ) were 50 μg/L for imatinib and nilotinib and 2. This system was found to give compact spots for dasatinib after development (R F value of 0. specificity. the drug was extracted with 0. 25 cm × 4. The first method was based on high performance thin layer chromatography (HPTLC) followed by densitometric measurements of their spots at 280 nm. with correlation coefficient values higher than 0. The collected extract (1 μL) was injected onto a Phenomenex Kinetex 50 mm × 2. The method proved to be accurate (% bias < 13. v/v).5–250 μg/L for dasatinib. As the proposed analytical methods could effectively separate the drug from its degradation products.[33] studied Imatinib. The second method was based on high performance liquid chromatography (HPLC) of the drug from its degradation products on reversed phase. D. 59 . After the addition of isotopically labelled internal standard. dry heat and photo-degradation conditions. v/v) pH 3.1 mm C18 column and eluted with acetonitrile gradient into a triple quadrupole ESI–MS/MS Agilent 6460 operated in positive mode. Eva Karlj et al. they can be employed as stability indicating.0 and retention time (t R = 8. dasatinib and nilotinib are three tyrosine kinase inhibitors currently used to treat Bcr-Abl1 positive chronic myelogenous leukaemia (CML). oxidation. The total run time was only 2.23 ± 0. Both separation methods were validated as per the ICH guidelines. selectivity. wet heat. Dasatinib was subjected to acid–alkali hydrolysis.Mhaske et al.V. precision. The drug was susceptible to acid–alkali hydrolysis and oxidation.23 ± 0.9.0. The method was linear in the range of 50–5000 μg/L for imatinib and nilotinib and in the range of 2. The separation was on HPTLC aluminium sheets of silica gel 60 F254 using toluene:chloroform (7.

In order to overcome insufficient sensitivity of dasatinib. The limits of quantification were in the range 1. Chromatography was performed on a Waters Acquity-UPLC® system with BEH C18-50*2. Samples were prepared by solid phase extraction (Oasis® MCX μElution) and eluate was injected in the system.51 min) were adequately separated. K. imatinib.[36] Tyrosine Kinase Inhibitors (TKIs) are a class of targeted drugs for the treatment of malignant pathologies.and inter day coefficients of variation were ≤ 15% and the chromatographic run time was 12 min.9% and the accuracy of the quality control samples ranged between 99. nilotinib. 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.11.5 ng/ml. sorafenib. nilotinib. under a gradient of ammonium formate–acetonitrile.[34] studied A simultaneous test for six TKIs (erlotinib.99 for all analytes.1 mm column. Peaks of each compound (retention time from 0.0–5. its metabolite.Calibration curves ranged from 10 to 5000 ng/mL for imatinib. An Acquity-TQD® with electrospray ionization was used for detection. lapatinib. We developed a fast method with analysis time of 55 s and 19 s in multiple injection setting.[35] studied Therapeutic drug monitoring is recommended for the optimal several malignant diseases.0 and 107. The mean relative extraction recovery was in the range of 90.76 to 2. 13.1 to 200 ng/mL for dasatinib. 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. multiple reaction monitoring cube mode in linear ion trap (MRM3) was successfully applied. The plasma for analysis was deproteinated by methanol and the centrifuged supernatant was directly injected to mass spectrometer without separation step. The intra. lapatinib. erlotinib and sorafenib and from 0. gefitinib and sunitinib. Lutz Götze et al.The calibration range was 10–1000 ng/mL for sunitinib and 50–5000 ng/mL for the other TKIs with coefficients of determination ≥ 0. The method was successfully validated and applied to the patient plasma samples. Micova et al.9%. axitinib. 60 . Stephane Bouchate et al.3–106.5%. sunitinib) was developed using liquid chromatography tandem mass spectrometry in a multiple reaction monitoring mode. Imprecisions were lower than 6. 12.

after correcting for blood to plasma ratio. and LC-tandem mass spectrometry method. the availability of limited samples may be a concern for multiple injections. an opportunity exists for the automated collection of rat PK time points using DBS. the feasibility of using DBS samples for drug metabolite profiling including both phase I and phase II metabolites has not been well established. Overall. The results obtained from this study suggest that collection of study samples by DBS can be used for metabolite profiling. Zhongzhou Shen et al. automated sampling of blood time points are routinely employed. 61 . HPLC-MS/MS. 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.14. 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. With thedevelopment of dried blood spot (DBS) technology for drug level quantitation. This work reports the study of metabolite profiling of dasatinibdosed to Wistar Han rats using automated DBS collection. DBS. Both phase I oxidative metabolites and phase II glucuronide conjugates and sulfate conjugates were detected from both rat plasma and DBS samples. Chemically treated DBS cards such as DMPK-A and DMPK-B cards may affect the dasatinibmetabolites. HPTLC. On literature survey it was found that. 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. Dasatinib and its metabolites were analyzed by multiple reaction monitoring (MRM) and MRM trigger enhanced product ion scan (MRM-EPI). Similar PK parameters were obtained for dasatinib from both plasma and DBS samples. Sweden) was employed using dasatinib as a model compound. However. Automated DBS and plasma collection using a rat AccuSampler (VeruTech AB. LC-MS. 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). however.[37] To characterize and enable efficient rat pharmacokinetic (PK) screening in early drug discovery.

one HPLC and spectrophotometric methods were developed and validated for the determination of title drug. linearity. These methods provide means to simultaneously characterize and quantify the components of a mixture without prior separation and derivatization. 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.In view of the need for a suitable method for routine analysis in formulation. attempts are being made to develop and validate simple. and system suitability. These proposed methods are suitable for the analysis in pharmaceutical quality control laboratories. 62 . The methods were validated for parameters like accuracy. Validation of the method was done in accordance with USP and ICH guideline for the assay of active ingredients. specificity. The utility of the developed method to determine the content of drug in commercial tablet is also demonstrated. precision.

To estimate the different compounds with less time in different formulations like tablets. Utilisation of minimum solvent.2.3. To reduce the run time as compared with previously reported literature.acidic and neutral analytes. Advantages of less run time in HPLC: It’s beneficial to the company economically. expectorants and injections. machine and materials. syrups. The reasons for developing RP-HPLC method for determination of the drug in tablet dosage forms are as follows: 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. Less utilisation of men. Trials were done on different coloumns and the optimized one is Cosmicil BDS coloumn. coloumns. capsules.1. As RP-HPLC was chosen as the instrument wide variety of equipment. Reduce the cost. 63 . 2.3. To develop a method for drug in its dosage form. 2. 3. eluent and operational parameters are involved in it.      To develop newer RP-HPLC Method by Isocratic Mode. Cosmicil BDS coloumn:[37]  BDS is a base deactivated silanol in which the residual silanol groups are deactivated which is suitable for the basic. SELECTION OF INSTRUMENT Dasatinib is polar in nature the RP-HPLC method was preferred because of its simplicity and suitability.

3. 2. Minimize the absorbance of buffer.0ml/min.2. Considering the above points methanol and acetonitrile are used as mobile phase . The drug must be stable in moile phase for atleast duration of analysis.Excesive salt concentration must be avoided which otherwise lead to damage of the equipment.2.e.    phosphate..potassium dihydrogen These coloumns shows wide pH range i. Mobile Phase: Mobile phases used for HPLC are typically mixtures of organic solvents and waters or aqueous buffers.3. The UV-detection range is 220-330nm. Methanol:[38]    It is also known as methyl alcohol.disodium phosphate.water. This coloumn is suitable for elution hydrogen with different eluents like methanol.6 Particle (µm) 5 Size C-18 BDS 2.1. It is a polar mobile phase which is used for RP-HPLC. 2-9. 2 . The UV-cutoff range 205nm. acetic acid etc. The flow-rate is 0.8-2. These are chosen based on the following points: 1. Column Dimensions  Type Length (mm) 150 Width (mm) 4.3.acetonitrile. 64 .

as little as 10 mL of pure methanol can cause permanent blindness by destruction of the optic nerve.2. although the median lethal dose is typically 100 mL (4 fl oz) (i. and also causing metabolic acidosis. First. 1–2 mL/kg of pure methanol. Formate is toxic because it inhibits mitochondrial cytochrome c oxidase. The conversion to formate via ALDH proceeds completely. 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. 2. Toxic effects take hours to start.e. for example. Acetonitrile:[39]   It is also known as methyl cyanide Its low viscosity and low chemical reactivity make it a popular choice for liquid chromatography. or absorption through theskin) can be fatal due to its CNS depressant properties in the same manner as ethanol poisoning. and effective antidotes can often prevent permanent damage. If ingested. with no detectable formaldehyde remaining. Methanol is toxic by two mechanisms.Toxicity: Methanol has a high toxicity in humans.3. Methanol is converted to formaldehyde via alcohol dehydrogenase (ADH) and formaldehyde is converted to formic acid (formate) via aldehyde dehydrogenase (ALDH). 65 .2. 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). Second. inhalation. in a process of toxication. causing the symptoms of hypoxia at the cellular level. methanol (whether it enters the body by ingestion. and 30 mL is potentially fatal. among a variety of other metabolic disturbances.

which do not usually appear for several hours after the exposure. Acetonitrile poisoning in humans (or. include breathing difficulties. 66 . with oxygen.Toxicity: Acetonitrile has only a modest toxicity in small doses. followed by death from respiratory failure. Generally the onset of toxic effects is delayed.[13] The symptoms. to be more specific. due to the time required for the body to metabolize acetonitrile to cyanide (generally about 2–12 hours). by inhalation. and vomiting: Convulsions and coma can occur in serious cases. which is the source of the observed toxic effects. slow pulse rate. sodium nitrite. It can be metabolised to produce hydrogen cyanide. ingestion and (possibly) by skin absorption. of cyanide poisoning after exposure to acetonitrile) are rare but not unknown. nausea. The treatment is as for cyanide poisoning. and sodium thiosulfate among the most commonly-used remedies.

1. 2. 2. 4. 1. CHEMICALS AND INSTRUMENTS 3. Ltd. Ltd. 5. 67 .1. Drug: S. Chemical name Acetonitrile Methanol Purfied Water Triethylamine Orthophosporic acid Grade HPLC HPLC MilliQ HPLC HPLC HPLC B.3. NATCO Pharma Pvt. 3. Drug name Dasatini b(std) Dasatini b(sample) Manufactured by NATCO Pharma Pvt. No. 1. Chemicals used: S.1 MATERIALS: A. No.

2. (150X4. BV .6mm) 2. Name of the instrument HPLC Column Waters Cosmicsil BDS C-18.40 Make 68 . 4. 3. No..5microns. Orbital shaker Analytical balance Membrane filter pH meter Centrifuge apparatus Afcoset Smart Labtech Pvt. 1.3.1. Instruments used S. 5. 6. Ltd.

60 µg/ml respectively with mobile phase. 5.40.2.0 ml was transferred into a 10 ml volumetric flask and made up to the mark to produce 20.50. MATERIALS AND METHODS 3.2. Cool the solution to room temperature and dilute to volume with solvent mixture. Solvent mixture:  Prepare a mixture of methanol and acetonitrile in the ratio of 50:50 v/v respectively. Transfer 1.2.30.0 ml .3.    Add about 30ml of solvent mixture and sonicate to dissolve.0 ml. 3.2.0ml of triethylamine to 100ml water and adjut the pH to 6. Mobile phase:  Prepare a filtered and degassed mixture of buffer and solvent in the ratio of 50:50 v/v respectively. 69 . 3.3.05 diluted with orthophosphoric acid add 10ml of methanol and mix well. 3.0ml of the above solution into a 10ml volumetric flask and dilute to volume with mobile phase.4.0 mg of Dasatinib monohydrate (working standard) into 50ml volumetric flask .4. 2.2.5±0.2.1. 3.5.0 ml and 6.0 ml.2. Preparation of working standard solution:  From the standard stock solution. Buffer preparation:  Add 4. Preparation of standard solution:  Accurately weighed quantity of the drug was transfered about 68. 3.

dilute to volume with mobile phase. Transfer 1ml of the above solution into a 10ml volumetric flask.  Add about 60ml of solvent mixture . Transfer 1ml of the above solution into a 100ml volumetric flask. shake on orbital shaker for 15min and sonicate for 30min with occasional shakings. Transfer an accurately weighed portion of powder. equivalent to 68.2.    Cool the solution to room temperature and dilute to volume with solvent mixture.  Add about 60ml of solvent mixture . 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. 70 .7. Centrifuge the solution at 3000RPM for 15min.3.0mg of Dasatinib into a 100ml volumetric flask.2.    Cool the solution to room temperature and dilute to volume with solvent mixture. shake on orbital shaker for 15min and sonicate for 30min with occasional shakings. dilute to volume with mobile phase. Preparation of sample solution:   Weigh and finely powder not fewer than 10 tablets. 3.6. Centrifuge the solution at 3000RPM for 15min.

METHOD DEVELOPMENT The objective of this study was to optimize the assay method for estimation of Dasatinib based on the literature survey made.2 ml / min : Devosil ODS C18. The chromatogram for trial-1 was shown in . Mobile phase was pumped at a flow rate of 1.6 mm. 5 m : : : : 315 nm 35 0C 10 l 10 min Mobile phase Solution of phosphate buffer (pH-6. 71 . Trial-1 Chromatographic conditions Flow rate Column Detector wavelength Column temperature Injection volume Run time : 1.3.2ml/min. acetonitrile and methanol in the ratio of 50:50 v/v is used. Observation The tailing factor of the peaks obtained was high & fails the system suitability. 150 mm X 4.3.5).

acetonitrile and methanol in the ratio of 60:40v/v at a flow rate of 1. 5 m column was used. Trail-2 Chromatographic conditions Flow rate Column Detector wavelength Column temperature Injection volume Run time : 1. 150 mm X 4. 5 m : : : : 311 nm 35 0C 10 l 10 min Mobile phase Solution of phosphate buffer (pH-6. The column temperature was maintained at 35 c. Cosmicsil ODS C-18.0ml/min.6 mm.5). 72 .Fig:2 Trial -1 chromatograph .2 ml / min : Cosmicsil ODS C18. 150 mm X 4.6 mm.

150 mm X 4.0 ml / min Cosmicsil BDS.6 mm. Fig:3 Trial -2 chromatograph Trial -3 Chromatographic conditions Flow rate Column Detector wavelength Column temperature Injection volume Run time : : : : : : 1. The chromatogram for trial-2 was shown. C18.Observation The tailing factor of the peaks obtained was high & fails the system suitability. 5 m 315 nm 25 0C 10 l 10 min 73 .

Cosmicsil BDS C-18. 150 mm X 4. 74 .5). OPTIMIZED METHOD FOR ASSAY Buffer preparation: Add 4.0ml of triethylamine to 100ml water and adjut the pH to 6.Mobile phase Solution of phosphate buffer (pH-6.46 min. 5 m column was used.3. The chromatogram for trial -3 (optimized method) was shown.05 diluted with orthophosphoric acid add 10ml of methanol and mix well.6 mm. Observation Peak of Dasatinib was well resolved with the retention time of 6. The column temperature was maintained at 25 c.0ml/min.1. Fig:4 Trial-3 chromatograph 3. Solvent mixture: Prepare a mixture of methanol and acetonitrile in the ratio of 50:50 v/v respectively.5±0. acetonitrile and methanol in the ratio of 50:50v/v at a flow rate of 1.

Add about 60ml of solvent mixture .0 ml was transferred into a 10 ml volumetric flask and made up to the mark to produce 20. 3. 5.0 ml.Transfer 1. 75 .Cool the solution to room temperature and dilute to volume with solvent mixture.40. Cool the solution to room temperature and dilute to volume with solvent mixture. dilute to volume with mobile phase.Mobile phase: Prepare a filtered and degassed mixture of buffer and solvent in the ratio of 50:50 v/v respectively.30.0 ml and 6.60 µg/ml respectively with mobile phase.0 ml .0ml of the above solution into a 10ml volumetric flask and dilute to volume with mobile phase. Centrifuge the solution at 3000RPM for 15min. Preparation of standard solution: Accurately weighed quantity of the drug was transfered about 68.Transfer 1ml of the above solution into a 10ml volumetric flask.Add about 30ml of solvent mixture and sonicate to dissolve. shake on orbital shaker for 15min and sonicate for 30min with occasional shakings.50.0mg of Dasatinib into a 100ml volumetric flask. Preparation of sample solution: Weigh and finely powder not fewer than 10 tablets. Transfer an accurately weighed portion of powder. equivalent to 68. Preparation of working standard solution: From the standard stock solution.4. 2.0 mg of Dasatinib monohydrate (working standard) into 50ml volumetric flask .0 ml.

blank and placebo preparations in duplicate were injected separately into HPLC system and the peak responses for Dasatinib were measured. 5 m 315 nm 25 0C 10 l 10 min 6. C18. Add about 60ml of solvent mixture .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.6 mm. Test Procedure 10 µl of the standard. shake on orbital shaker for 15min and sonicate for 30min with occasional shakings. 150 mm X 4. : : : : : : : 1. sample.467 76 .0 ml / min Cosmicsil BDS. The quantities from the peak area in mg of Dasatinib were calculated per tablet taken.Transfer 1ml of the above solution into a 100ml volumetric flask. 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. Centrifuge the solution at 3000RPM for 15min. dilute to volume with mobile phase. Cool the solution to room temperature and dilute to volume with solvent mixture.

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 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 77 .x ----------x --------.

33 311297 311287 311277 311307 311267 311259 311282.8 78 .33 0.04889 50 99. 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.6 50 99.purity Sample weight Label amount %Assay 311353 311363 311343 311323 311345 311333 311343.Table 3.8 489.1.

Precision 5.1.24 7620 79 . Table 3. Linearity 3. 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. 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. LOD & LOQ 3. System suitability 2. of theoretical plates Mirtazapine 0.78 % 1. The following parameters have been validated. 1.4.2. Accuracy 4. System suitability data by HPLC System suitability Parameters %RSD Tailing factor No.4.3. Robustness 6.

The linearity of the calibration graph was validated by the high value of correlation coefficient. 3.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. slope and the intercept value was shown 80 . In this study Six concentrations were chosen ranging between 20-60µg mL-1 for Dasatinib.4. 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 . Linearity: Linearity of the proposed HPLC method for determination of Dasatinib were evaluated by analysing a series of different concentrations of standard drug.0.0.  The tailing factor for Dasatinib should not be morethan 2.2. From the system suitability studies it was observed that all the parameters were within limit.

1 2 3 4 5 Concentration (µg / 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. 81 .999. Linearity range and average area values Solution No.Table:3.3.999 Calibration curve of Dasatinib Acceptance criteria Correlation coefficient should be not less than 0.

The repeatability (within-day in triplicates) and intermediate precision (for 3 days) were carried out at six concentration levels for compound. 82 .3. The precision expressed as % RSD is given .4. Precision Precision of the analytical method was studied by analysis of multiple sampling of homogeneous sample.4.999 3. It was demonstrated by repeatability and intermediate precision measurements of peak area and peak symmetry parameters of HPLC method for the title ingredient.999 99. Triplicate injections were made and the obtained results within and between the days of trials were in acceptable range.Table 3. Calibration parameters for Dasatinib Parameter Results Slope Intercept Correlation co-efficient Percentage curve fitting 7985 -11196 0.9% Observation The linearity Correlation coefficient for dasatinib is 0.

Observation The Relative standard deviation was found to be 0.78 % RSD* *ˉaverage of 6 replicate injections for each concentration Acceptance criteria Relative standard deviation of percentage assay results should not be more than 2.78% .5. of dasatinib Peak Area (g/mL) 309400 312247 305016 40 307219 307467 308121 0. Table 3.3. Repeatability Six sample solutions were prepared and injected into the HPLC system as per test procedure.1.3.4. 83 . Results of repeatability Conc.0 %.

Table 3.4. Accuracy Accuracy of an analytical method is the closeness of test results obtained by that method to the true value. 3. Intermediate precession (analyst to analyst variability) Two analysts as per test method conducted the study.4. Observation The Relative standard deviation was found to be 0.2.80 Acceptance criteria Relative standard deviation of % assay results should not more than 2. For Analyst-1 refer precision (Repeatability) results and the results for Analyst-2 were discussed below.0 % by both the analysts. Accuracy was performed in three different levels. The accuracy of an analytical method should be established across its linearity range.3.80% . Results of intermediate precession Conc. each level in triplicate for 84 . of dasatinib (g/mL) 308344 307467 307219 50 305017 312247 309402 *-average RSD of 6 replicate injections Peak Area % RSD* 0.3.6.4.

0 99.2 100.Each sample was analysed in triplicate for each level.0.7 100.99.247 %RSD.16 S.D.151 Mean. 3. 100% and 150%.26 S.15 49.1. 3.96 S. 2. Table 3. 2.0 %.247 1. 2.90 100.D.20 150. 3. Acceptance criteria The mean % recovery of the Dasatinib monohydrate at each spike level should be not less than 98.3 99.85 150. 1.0 149. Observation: The mean % recovery levels were found to be 100.0. 1.19 Mean.8 100.109 %RSD.0.1 100.20 50.100. 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 50.0.0.D.4 100.108 Mean.7. 85 .Capecitabine using standards at 50%.152 %RSD.0.50 100.09 100.100.0 % and not more than 102.5 100.10 100.

Chromatogram of blank should not show any peak at the retention time of analyte peak.3. Fig 6 Standard chromatogram for Dasatinib identification 86 . Solutions of standard and Sample are prepared as per test method and injected into the chromatographic system. Specificity Specificity is the ability to asses unequivocally the analyte in the presence of components which may be expected to be present. Mobile phase was injected as per the test method.Lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedures.5.4. Blank interference: A study to establish the interference of blank was conducted.

Fig 7 Chromatogram for blank interference Fig 8Chromatogram for placebo interference 87 .

27 and 35ºC temperature.0 ml/min and 1. Effect of variation of temperature A study was conducted to determine the effect of variation in temperature. 88 . Standard solution prepared as per the test method was injected into the HPLC system at 25.4.2. 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. The effect of variation of flow rate was evaluated.6.4.6.3.2 ml/min.1.4. 3. 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 1.6. 3.  The system suitability parameters were evaluated and found to be within the limits for a temperature changes.

3 σ / S = 3.3 (98.0405 µg / ml.0405 Detection limit was 0.Table 3.1936 / 7985) = 0. 89 . Limit of detection (LOD) Calibration curve was repeated for 5 times and the standard deviation (SD) of the intercepts was calculated.1.2 increased and at higher flow rates the relative Ambient in symmetry.Results of robustness Optimum Parameters range procedure At lower flow rates the asymmetry factor was Flow ml/min retentions was decreased.8.2 1.4. Beyond the optimum range there is a change Temperature 25. The LOD was determined by the formula: LOD = 3.35 C o Conditions in Remarks rate 1.7. 3.0.

90 . 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 σ / S = 10 (98.1229 Quantification limit was 0.1229 µg / ml.4.1936 / 7985) = 0.8.3.

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. The low values of % R. simple.S. which were prepared and analyzed on same day. tailing. intercept -11196 and slope 7985 for Dasatinib monohydrate . run time.D. The % R. The calibration was linear in concentration range of 20-60 µg mL-1 with regression 0.D indicate the method is precise and accurate. Mobile phase and flow rate selection was based on peak parameters (height.9999. In the method development. The retention time for Dasatinib monohydrate was found to be 6.05):solvent mixture [acetonitrile: methanol(50:50 v/v)] in the ratio of 50:50v /v is used gave a better resolution and sensitivity. theoretical plates. an adequate separation of eluted compound.S. Sample to sample precision and accuracy were evaluated using t samples of different concentrations. These results show the accuracy and repeatibility of the assay. capacity or symmetry factor). 91 .The proposed method was validated in accordance with ICH parameters and the applied for analysis of the same in laboratory prepared mixtures.467 min . accurate and economic and then applied on pharmaceutical dosage form. Various ratios of mobile phase systems were prepared and used to provide an appropriate of of buffer (pH-6. reported was found to be less than 2 %. HPLC conditions were optimized to obtain.5±0. precise. The optimum wavelength for detection was 315 nm at which better detector response for the title drug was obtained.

The proposed methods are accurate.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. rapid and selective for the estimation of Dasatinib monohydrate in laboratory prepared mixtures. 92 . simple. Limit of Quantification and Limit of Detection were found to be 0.0405 µg / ml respectively. Hence. these methods can be conveniently adopted for the routine analysis of Dasatinib monohydrate in quality control laboratories.1229 µg / ml and 0.

SUMMARY & CONCLUSION A HPLC method was developed for the estimation of Dasatinib in tablet dosage form using reverse phase high performance liquid chromatography.18 with ambient temperature. Hence the proposed method was found to be satisfactory and could be used for the routine analysis of Dasatinib in the tablets.5).The proposed method was applied for the determination of Dasatinib in tablets. ruggedness and robustness. injection volume of 10µl is injected and eluted with mobile phase of phosphate buffer (pH-6.4675min. The peak of Dasatinib was found at 6. which was pumped with a flow rate 1.No:2690) with UV\VIS detector and Cosmicsil BDS C.The developed method was validated for various parameters as per ICH guidelines like accuracy. Acetonitrile and methanol in the ratio 50:50 v/v.HPLC Waters (Model. LOQ. 93 .0ml/min and detected by UV at 315nm. precision. linearity. LOD.

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