CHAPTER - 1 INTRODUCTION. Analytical methods development, identification [1], characterization

CHAPTER - 1 INTRODUCTION Analytical methods development, identification [1], characterization [2] of impurities [3] and method validation play key role in the pharmaceuticals discovery, development, and manufacturing. The new drugs introduced into the market are increasing every year in number. These drugs may be totally new or partial structural modification in existing molecules. Based on reports of toxicities and continuous wider use of these drugs are indicated. Introduction of its better replacement, these new drugs may include later in official pharmacopoeia. Therefore, due to this lag time, analytical procedures, and standards may not available in concerned pharmacopia, we can not find these drugs and impurities even the drug substances [4] and drug products introduced into the market are increasing every year. These drug substances and drug products may be either partial structural modification of the existing drug substances or new entities. This happens because of continuous and wider usage of these drug substances and drug products and possible uncertainties, introduction of better drugs by competitors and reports of impurity toxicities. Under these circumstances, analytical procedures and standards for these drugs and their related substances [5] may not be available in the pharmacopoeias eg. Indian, IP [6], United Kingdom, BP 1

[7], United States, USP [8], European, EP [9], Japan, JP [10] and Martindale Extra [11] and public domain. Combination products referred as with more than one drug (Fixed dose combinations), previously not available to the patients by combining theraputic effects of two or more drugs in one product. In such cases, development of new analytical methods plays an important role. Study drugs including structure, chemical nature, and synthesis composition need to make use of science in the aspect of bio-chemical actions are part of pharmaceutical chemistry [14-21]. Influence on an organism and studies the physical and chemical properties, the methods of quality control and conditions of their storage. India is the richest source for Pharmaceutical products. Developed countries like United States of America, Australia and Britain etc. have much demand for these medicines; hence India is utilizing this opportunity to promote the products. Presently we are exporting formulations to many developed countries, but these countries are now imposing their regulatory guidelines to accept our products into their markets. Australia is now regulating manufactures by TGA [22] Therapeutic goods administration, America by USFDA [23] United States Food and Drug Administration and European regulatory agency by EMEA [24], Brazil by ANVISA [25] and other countries by ICH [26] and WHO [27]. According to these stipulations the therapeutic products must satisfy defined specifications like product description, identification, and assay for active 2

constituents, related substances or degradation products, microbial purity, and stability data for the exported product through out its shelf life. Indian manufacturers require analytical techniques and tools to define the quality of their products and to retain their qualification in world markets. The analytical tools include chemical, physico-chemical, instrumental, biological techniques and also the combination of both with instrumental methods (hyphenated techniques [28]) for developing qualitative and quantitative determinations. Analytical chemistry plays an imperative role to success the progression. 1.1 An Introduction to Pharmaceutical Formulations: A pharmaceutical drug can be broadly defined as any chemical substance intended for use in the medical diagnosis, cure, treatment, or prevention of disease, also referred to as medicine, medicament or medication [29-31]. Pharmaceutical formulation is a produce of final medicinal product by process of different chemical substances, including the active drug are combined [32]. Pre-formulation, study has to be done on behavior of a given steroid and antibiotic under a variety of stress conditions such as freeze/thaw, temperature, shear stress among others to identify mechanisms of degradation and therefore its mitigation [33]. 3

1.2 Types of pharmaceutical Formulations: The drugs used in various forms in prophylactic or in therapeutic [34-37] use. They are formulated as tablets, capsules, dry syrups, liquid orals, creams or ointments, parenterals (injection in dry or liquid form), lotions, dusting powders, aerosols etc. In tablets one or more among the diluents such as starch, lactose, cellulose derivatives, calcium phosphate, mannitol, sorbitol, sucrose, aerosols, acacia, polyvinyl pyrrollidine, alginic acid, tragacanth, stearic acid, talc, magnesium stearate, waxes, methyl paraben, sodium benzoate, permitted flavours, and colors may be added. In capsules one or more among the diluents, certified dyes, gelatine, plasticizers, preservatives, starch, lactose, talc may be added. In dry syrups and liquid orals, sucrose, sorbitols, preservatives, certified colors, and flavours might be added. In creams and ointments, waxes, carbopol, petroleum jelly, surfactants, preservatives permitted colors, and perfumes might be added. In parenterals, water, vegetable oils, mineral oils, simulated oils, propylene glycol, dioxalamines, dimethyl acetamide may be used as vehicles. Any one or more among stabilizers, anti-oxidants, buffering agents like citrates, acetates, phosphates, co-solvents, wetting suspending and emulsifying agents like tween-80, sorbitol, oleate and preservatives may be added. In lotions, dusting powders and aerosols, talc, silica derivatives, alcohol, preservatives may be added. 4

In some instances drugs are applied in small doses and they are often mixed with excipients as combinations. The assay of various dosage forms raises several special and skilful sampling for the preparation of sample solutions. Hence standard techniques must be employed to ascertain the homogeneity of the sample before collecting for analysis. 1.3 Preparation of Sample Solution for Analytical Investigations: Preparation of sample solution may not always a straightforward situation. Preparation often includes processes such as quantitative extraction frequently causes serious problem, extraction of drugs into the most important solvents and their nature to be bound to excipients, each case it can be solved seperately. The most difficult problems arise when selective extraction is necessary. It is also observed often that the specificity of the extraction is insufficient. In these instances separation of the components by extractor and its further chromatography are widely used for purification. The most exact method of extracting drugs from formulation is to treat the drugs with a proper solvent, RS method chosen such that the resulting extract can be used directly in the assay without having the intereference of associating ingredients. When the finely pulverized sample is agitated and/ or assisted with boiling the solvent for a period from few minutes to several hours to get sufficient extraction achieved. In general, a sample later used as is or concentrated well, if the content is very less to improve the sensitivity of the analysis. 5

Increasing the selectivity of the two-phase extraction is generally used method for decreasing the absorption loses. Here one of the solvents is always water. Water soluble components such as the generally used method for decreasing the adsorption losses and increasing the selectivity of the extraction are two-phase extraction. Here one of the solvents is always water. Lactose, is generally main ingradient of the excipients to get favorable conditions for extraction of the drugs and their related substances. Starch is also critical from the point of view of adsorption losses, which can be eliminated by dissolving treatment with diastase and other solvents, which are immiscible with water. The organic solvents genereally used dichloromethane, chloroform, ethyl acetate, diethyl ether, iso-octane and few others have also been used. 1.4 Introduction to Analytical Chemistry [38]: Analytical chemistry is a science deals with the identification, characterization and estimation of the components of a sample. The primary interest of an analytical chemist is to develop experimental methods of measurement to obtain information about the qualitative and quantitative tests for given composition of a sample. Analytical chemistry involves a multisided approach to obtain information of every individual chemical species present in any sample. A knowledge of analytical chemistry helps to develop methods, select an appropriate instruments, and strategies to obtain information on the composition and nature of sample. The number of analytical techniques, their degree of 6

sophistication and area of applications has increased tremendrously [39-40]. Analytical chemistry [41] as a whole has evolved world wide supporting pillar of human culture, industry and trade, providing numerous goods of urgent daily need to the human kind, such as pharmaceuticals, chemicals and also food ingredients. Analytical chemistry stands in the development of science [42]. The most important definition of analytical chemistry was proposed by the working committee on analytical chemistry of the FECS [43] (Freedom of European Chemical Societies). It reads all branches of chemistry, and techniques drawn on the ideas of analytical chemistry [44-45]. Analytical chemists [46] meet the demands for better chemical measurements reliability of existing techniques to be improved. Analytical chemist efforts to develop new methods those methods developed are kept purposely static; so that data can be compared over long periods of time and this is true in industrial quality assurance (QA). In Analytical chemistry the use of a tunable laser to increase the sensitivity and specificity of a spectrometric method plays an important role in the pharmaceutical industry, where aside from QA [47], it is used in discovery of new drug candidates. Analytical chemistry has been important because Modern analytical chemistry [48] categorized in terms of the analytical target, in providing methods for determining elements and chemicals are present in the world around us. The analytical methods and their research applications [49-54] are Material analysis, Bio-analytical chemistry, Chemical analysis and 7

Forensic chemistry. Analytical methods are differntiated as wet-chemical and instrumental methods. Wet-chemical methods include gravimetric and volumetric analysis while instrumental methods include mass spectrometry [55], spectrophotometry [56-58] and colorimetry, electrophoresis, crystallography, microscopy, electrochemistry, optical methods such as emission and absorption spectroscopy etc., separation methods such as chromatography, electro analytical methods like potentiometry, voltammetry etc., radiochemical methods like scintillation, tomography and biological methods like RIA and ELISA tests [59]. Analytical techniques [60-63] are broadly classified as titration techniques, spectrometric techniques and chromatographic separation techniques. Different type of detectors [64] used in chromatographic technique are Photo Diode Array detector, Light scatering Evaporative detector, Refractive index detector, Ultraviolet detectors, Mass detectors, Potentiometric detectors, Fluorascence detectors, Amperometric electrochemical detectors, Flame ionization detectors, Thermal conductivity detectors, Electro chemical detectors. Sophisticated instrumentation given predominant status to Modern Analytical chemistry, the basic roots of analytical chemistry and some of the methods used in advanced instruments are from titration [65-66], gravimetry [67], inorganic qualitative analysis [68-69], crystallography [70-71], electrochemical analysis [72], thermal analysis [73], separation techniques [74], hybrid techniques [75-77], microscopy [78], and 8

traditional analytical techniques [79-81]. Chromatographic separation techniques are multi-stage separation methods. In two phases, we can find the components of the sample, one is stationary and another one is mobile. The stationary phase prepared is a solid or a liquid supported on a solid or a gel. The stationary phase is packed in a column, spread as a layer, or distributed as a film, the mobile phase may be gaseous or liquid or supercritical fluid. The separation may be based on adsorption, mass distribution (partition), ion exchange, etc., or may be based on differences in the physico-chemical properties of the molecules such as size, mass, volume, etc. chromatographic separation methods are used for simulltanious estimation of combination drugs. These methods offer accuracy and precision and good reproducibility. Chromatography classification [82-85] is based on its interaction with the stationary phase. 1.5 High Performance Liquid Chromatography [86-110]: High Performance Liquid Chromatography (HPLC) is a different branch of column chromatography in which the mobile phase is forced through the column at high speed. As a result the analysis time is decreased by 1-2 orders of magnitude relative to classical column chromatography and the use of much smaller particles of the adsorbent or support becomes possible increasing the column efficiency substantially. 9

HPLC General Considerations [111-115] involving a polar stationary phase and a non polar mobile phase are indicated as normal phase systems and this combination of phases. Solute retention generally increases with solute polarity. High performance liquid chromatography comprises of a solvent delivery system or a pump for controlled flow of mobile phase and injection system for precise sample introduction. Variety of modified, stable chemically bonded stationary phases capable of being operated at high pressures, leading to better efficiency of separations. Sophisticated detector system capable of handling small flow rates and detecting small concentrations. The sample related information that needs to be known prior to HPLC method development. High performance liquid chromatography column is selected depending on the nature of the solute and the information about the analyte. Reversed phase mode of chromatography facilitates a wide range of columns like octadecaylsilane (C18), Octylsilane (C8), butyl silane (C4), phenyl, nitro, amino, cyanopropyl (CN), dimethyl silane (C2) etc. were chosen for this study since it is retentive, rugged and widely available. C18 and C8 columns are applied to approximately 80% of the reverse separation problems. The other available phases are often significantly less retentive than the C18 and C8 phases, and finding an appropriately 10

weak mobile phase that will accomplish the separation is not always possible due to the polar to semi polar nature of the analytes. Table 1.01 Column Particle Characteristics for HPLC Features Utility 5 μm totally porous particles Most separations 3 μm totally porous particles Fast separations 1.5 μm pellicular particles Veryfast separations (especially macromolecules) +50% from mean particle-size distribution 7 to 12 nm pores, 150 to 400 m 2 /g narrow pore 15 to 100nm pore, 10 to 150 m 2 /g wide pore Stable, reproducible, more efficient columns with lower pressure drop Small molecule separations Macromolecular separation. 11

Figure 1.01 Steps in HPLC Method Development Information on sample, define separation goal Need for special HPLC procedure sample pretreatment etc.? Choose detectors and detector settings Choose LC method; preliminary run; estimate best separation conditions Optimization separation conditions Check for problems or requirement for special procedure Recover purified material Quantitative Calibration Qualitative method Validate method for release to routine analysis 12

Figure 1.02 Method Development Conditions for the Initial Experiment. Nature of Sample HPLC CE GC SFC TLC Regular Special Neutral Ionic Inorganic ions Exploratory run Reverse-phase Isomers Enantiomers Isocratic Biological samples Gradient Ion-pair NARP Peptides Carbohydrates Nucleotides Macromolecules Normal-phase Proteins Nucleic acids Carbohydrates Synthetic polymers 13

Table 1.02 Column Selection for HPLC Method Development Reversed-phase and ion-pair method C18 (Octadecyl or ODS) C8 (Octyl) Rugged, highly retentive, widely available. Similar to C18 but slightly less retentive. C3, C4 Less retentive and mostly used for peptide and proteins C1[trimethylsilyl (TMS)] Phenyl, phenethyl CN (cyano) NH2 Amino Polystyrene Least retentive and least stable Moderately retentive and some selectivity differences Moderately retentive and used for both reversed and normal phases Weak retention, used for carbohydrates and less stable Stable with 1<pH<13 mobile phase and better peak shape and longer column life for some separations. Normal-phase method CN OH NH2 Silica Rugged and fairly polar general utility More polar than CN Highly polar and less stable. Very rugged and cheap, less convenient to operate, used in prep LC 14

Table 1.02 (Continued) Column Selection for HPLC Method Development Size-exclusion method Silica Silanized silica OH Polystyrene Very rugged and adsorptive Less adsorptive, wide solvent compatibility, used with organic solvents Less stable, used in aqueous SEC gel filtration Used widely for organic SEC (GPC), generally incompatible with water and highly polar organic solvents. Ion-exchange methods Bonded phase Less stable and reproducible Polystyrene Less efficient, stable, more reproducible. Mobile phase: Mobile phases in reversephase chromatography contains mixture of aqueous phase and organic phase, buffer is not required for neutral samples. Whenever acidic or basic samples are separated, it is strongly advisable to control mobile-phase ph by adding a buffer. It is strongly recommended that the ph of the buffer be adjusted before adding organic. In selecting buffer several considerations should be kept in mind. Buffer capacity is determined by ph, buffer pka, and buffer concentration. As for the case of sample compound, buffer ionization occurs over a range in ph given by pka + 1.5 and buffer can be effective only in this ph range. Buffers selected for a particular separation should 15

be used to control ph over a range pka +1 and concentration of 10 to 50 mm is usually adequate for reversed phase separation. A mobile phase with marginal buffer capacity will give less reproducible separation for compounds that are partially ionized at the ph of the mobile phase. In this case, retention may change from run to run, and distorted peaks may result. Table 1.03 Buffer Selection for HPLC Buffer pka Buffer Range UV Cutoff Trifluoracetic acid 2.0 1.5 to 2.5 210 nm Phosphoric acid/mono or di potassium phosphate 2.1, 7.2, 12.3 Less than 3.1 6.2 to 8.2 11.3 to 13.3 200 nm Citric acid/tri potassium citrate 3.1,4.7,5.4 2.1 to 6.4 230 nm Formic acid 3.8 2.8 to 4.8 210 nm Acetic acid 4.8 3.8 to 5.8 210 nm Mono /dibasic potassium carbonate Bis-tris propane HCl/ Bistris propane 6.4 5.4 to 7.4 Less than 200 nm 10.3 9.3 to 11.3 Less than 200 nm Tris HCl / Tris 8.3 7.3 to 8.3 205 nm Ammonium Ammonia chloride/ 9.2 8.2 to 10.2 200 nm 1-Methyl piperidine HCl/1- Methyl piperidine 10.1 9.1 to 11.1 215 nm Triethylamine Triethylamine HCl/ 11.0 10 to 12 200 nm 16

Buffer capacity is determined by ph, buffer pka, and buffer concentration. In case of sample compound, buffer ionization occurs over a range in ph given by pka + 1.5 and buffer can be effective only in this ph range. Buffers selected for a particular separation should be used to control ph over a range pka +1 and concentration of 10 to 50 mm is usually adequate for reversed phase separation. A mobile phase with marginal buffer capacity will give less reproducible separation for compounds that are partially ionized at the ph of the mobile phase. In this case, retention may change from run to run, and distorted peaks may result. UV absorbance of the selected buffer should exhibit as low as UV absorbance for proper detection, and to get good sensitive method. Retention in reversed phase chromatography affected by mobile phase composition such as choice of % B (organic), mobile-phase strength, column and temperature effects. Selectivity in reversed-phase chromatography influence factors such as solvent-strength selectivity, solvent-type selectivity, column type selectivity, temperature selectivity. An effective approach to method development begins with a very strong mobile phase. (e.g., 100% Acetonitrile) The initial use of strong mobile phase makes it likely that the run time of the first experiment will be conveniently short, and strongly retained compounds will all be 17

eluted. If no peaks observed after 30 to 40 min with 100% acetonitrile, weaker mobile phase is required, strength of acetonitrile gradually reduced to 80%, 60% which ever the amount until required separation and optimum run time attained. During HPLC method optimization stage, the initial sets of conditions that have evolved from the first stages of development are improved or maximized in terms of resolution, peak shape, plate counts, asymmetry, capacity, elution time, detection limits, limit of quantization, and overall ability to quantify the specific analyte of interest. When optimizing any method, an attempt should be made to provide analytical figures of merit which are needed to meet the assay requirements defined at the initial stages of method development. In other words, the required detection limits, limits of quantization, accuracy and precision of quantification, and specificity must be defined. Without adequate and definitive requirements, it is difficult to optimize any analytical method [116]. Two- columns strategy method used to enable rapid early method development along with a four-column method for commercial method development of the analytical methods utilized to evident the quality of drug substance or drug product. Mobile phases contain methanol or acetonitrile with aqueous trifluoroacetic acid for low ph screening and ammonium hydroxide for high ph screening [117]. 18

To determine the success of the method developed, HPLC column stability is one of the important factors. A systematic approach for the detection of HPLC column stability has been improved with emphasis on the usage of the application to pharmaceutical analysis. The specifics of the design, evaluation criteria mentioned and result obtained for some of the mostly used analytical columns from reputable column manufacturers. A stability profile over the ph range was identified that may serve as a reference for column scouting during method development [118] A brief study on the development of validated stability- indicating assay methods (SIAMs) for drug substances and products focused on critical issues related to method development of SIAMs. Frequent problems encoutered were in-sufficient impurity profiling and nonsuitability of pharmapoeial methods for the testing the stability samples [119]. The chromolith monolithic column indicates an efficiency that is comparable to that of columns compacted with spherical particles of 3 μm and 4 μm. We can compare the theoretical analysis speed of different seperation media by a kinetic plot analysis. At 200 bar, the monilith column indicated the highest performance when the requisit plate number was higher than 5000, while lower range, identified with compacted columns. If the possibility of optimum performance was used 19

the monolith column would provide the least efficiency while the column compacted with 1. 5 µm particles offered the shortest impedance time [120]. 1.6 Identification and Characterization of Impurities [121-122]: The United States Food and Drug Administration (FDA) have endorsed the guidance prepared under the auspices of the ICH. The guidance developed with the joint efforts of regulatory agencies and industry. Professionals from the European Union, Japan and the United State to ensure, that the different regions have consistent requirement for the data that should be submitted in the drug substance and drug products, in regards guidelines are not only aid the sponsors of latest drug applications (NDA) or abbreviated new drug applications (ANDA) with the type of information that should be submitted with their applications, but also assist the FDA reviewers of field investigators in their consist interpretation and implementation of regulations. FDA guidance on the impurities [123] for IND requires and maintain appropriate standards of identity, strength, quality and purity as needed for safety and give significance to clinical investigation made with the drug statement on the method, facilities and controls used for manufacturing, processing, and packing of new drug to establish NDA demands, more specific and explicit information, including stability studies to guarantee that all predetermined attributes of the drug product are maintained until its expiration date. The FDA may initiate 20

action under the F&D act to bring about removal of a product from the market, or as it granted in the law, a manufacturer may voluntarily withdraw from the market place any batches that do not meet the approved specifications. In each country regulatory authorities use their own standards for conducting clinical studies on a new drug product [124] and drug substance that has been formulated or the product that is approved for commerce. All efforts taken to characterize the source of actual impurities present in the new drug substance at a level greater than 0.1 per cent should be described. ICH guidelines also emphasize the characterizing the impurities present in the new drug products appearing as degradation products, identified in stability studies conducted on storage conditions at a level greater than the identification threshold (1 % for maximum daily dose of less than 1.0 mg and 0.1 % for a maximum daily dose of greater than 2 grams [125]. Developments in the field of magnetic resonance have been evidant strides in increasing sensitivity levels. This is most important in the structural characterization of drug impurities and degradation of products, which frequently are available only in extremely less quantities. The non-invasive nature of NMR spectroscopy makes it a valuable tool for the characterization of low level impurities and degradation products. In addition NMR can be considered close to a universal detector for hydrogen and carbon, as well as for other 21

magnetically active nuclei. This is both good and bad because all signals are detected, those arising from the compound of interest and all other compounds in the sample, such as solvents and starting materials. The popularity of LC-MS-MS systems for complex mixtures analysis of thermally liable biologically relevant molecules is largely attributed to the soft nature of atmospheric pressure ionization techniques such as electro spray ionization(esi), atmospheric pressure photo ionization(appi), attributes of various mass analyzers and scan modes used for collisioninduced dissociation experiments and issues. The next step generally is to obtain molecular mass and fragmentation data via HPLC_MS. It is essential to determine the molecular mass of the unknown, as it helps in tracking the correct peak by HPLC when isolation becomes necessary. To run LC-MS a mass spectrometry, a compatible HPLC method is necessary. If such a method is not available, it has to be developed which adds considerable time to the identification process. Relevance of Impurities characterization: The characterization effort serves as the basis of understanding for chemical and physiological process upon which successful development of a medicine depends. Traditionally, the product development process for pharmaceuticals has relied on separation techniques such as HPLC employing non specific detectors (refractive index detectors, UV/Vis absorption, electrochemical, fluorescence, etc.) These detectors provide sufficient information in many instances and are inexpensive, reproducible, rugged, and simple to 22

operate sample in the absence of authentic standards, they do not indicate any information that might lead to the recognition of compounds. In contrast, the use of a specific mode of detection, such as mass spectrometry alleviates the dependency on standards because compounds can be identified directly, provided they respond under the conditions of the analysis and that the molecular mass can be associated with the identification of particular compound. The most prevalent use of qualitative mass spectrometry during the course of pre clinical pharmaceutical development is structural elucidation of related substances (metabolites, synthetic impurities, and degradants), often at trace levels. Insight into chemical structure is a key factor in referring pharmaceutically desirable attributes of molecule such as potency, safety and bioavailability, thus elucidation of structure is critically important for accelerating the pace of the development process. The number of synthetic impurities and their levels are indicative of the overall process quality [126]. 1.7 Validation of Analytical Method [127-130]: Method validation constitutes the important part of any analytical methods. A Success in these areas is identified to several important factors, we can find in the development of pharmaceuticals and all of them have great contribution to regulatory compliance. Validation proves under standardised set of conditions that any procedure, process, equipment, material, activity or system performs; it assures that a 23

method works reproducibly, when proved by a same or different person, in same or different laboratories, using different reagents, different equipments, etc. Validation is important one to understand the parameters or characteristics involved in the validation process. Method validation is explained as the process of explained an analytical method acceptance in scientific method for its intended use. Guidelines for methods improvement and validation for noncompendial and compendial test methods are standerdised by the FDA and ICH documents, Analytical Procedures and Methods Validation: Chemistry, Manufacturing and Controls Documentation [104] and ICH topic Q2B. This latest document indicates to the method development and validation process for products included in investigational new drug (IND), new drug application (NDA) and briefed new drug application submissions [59]. In recent years, trails have been developed to the harmonization of pharmaceutical regulatory requirements in the United States, Europe, and Japan. The FDA methods validation draft guidance and USP refer to ICH guidelines [131]. 1.7.1 Accuracy: The accuracy of an analytical method indicates the similarity of agreement between the value, which is accepted either as a conventional 24

value or a proved reference value and the value identified. Accuracy is calculated as percentage recovery of the analyte by the assay of known added amount of the analyte in the sample. Accuracy was studied at four concentration level at LOQ level, 50% level, 100% level and 150% level of working concentration for related substances and 80% level, 100% level and 120% level for assay in triplicate. Each preparation was prepared independently by spiking impurities in to sample for related substances and analyte in the placebo for Assay. Percent of recovery was calculated by comparing values got in spiked sample with those obtained in standard and got results were recorded. 1.7.2 Precision: Precision can be defined as the degree of agreement among the individual test results when the procedure is applied repeatedly to multiple sampling of a homogeneous sample a more comprehensive definition proposed by International conference on Harmonization (ICH). Precision defined in to three types (1) repeatability (2) Intermediate precision and (3) reproducibility. To compare the system precision (repeatability) for peak response obtained with six replicates of dilute standard at limiting concentration. To check repeatability (method precision) of method for independent six different sample preparations were injected and % RSD with six sample 25

preparation found to be within 2.0%. To demonstrate Intermediate precision, the method be compared on two days on two different systems. 1.7.3 Linearity and Range: The linearity of the method is a measure of how well a calibration plot of response versus concentration approximates a straight line. To establish linear relationship between concentration and response with correlation coefficient will be more than 0.999. To propose best fitting equation of least square line (y = mx +c). 1.7.4 Limit of Detection: The lowest concentration of analyte that can be identified, but not able to determine in a quantitative method by using a specific method under the required experimental states. The instrumental method limit of detection determination is carried out by detecting the signal-to-noise ratio by comparing test values from the samples with known concentration of analyte with those of blank samples and the minimum level at which the analyte can be truly identified. A signal-to-noise ratio of 2:1 or 3:1 is standardised, the IUPAC approach gives the standard seperation of the intercept (Sa) which may be related to LOD and the slant of the calibration curve, b, by LOD = 3 Sa / b. 1.7.5 Limit of Quantitation: 26

Impurities in bulk drugs and degradation products in finished pharmaceuticals identified as per Limit of quantitation is a quantitative assay parameter for low levels of compounds in sample matrices. The limit of quantitation is the lowest determination concentration of analyte in a sample with standardised precision and accuracy when the standard procedure is applied. The standard deviation multiplied by a factor gives finalisation of the limit of quantitation. In many instances, approximately the limit of quantitation is double the limit of detection. 1.7.6 Specificity and Selectivity: The selectivity of an analytical method is its ability to measure accurately and especially the analyte of interest in the present of components that may be expected to be identify in the sample matrix. Chosen analytical method is capable to resolve and differentiate various items of a mixture and identify the analyte qualitatively. A method is said to be exact when it measures or proves quantitatively the component of interest in the sample matrix without separation. Hence primary deviation in the selectivity and speciality is that, while the previous one is restricted to qualitative detection of the components of a sample while the latter quantitative measurement of one or more analyte. Selectivity may be defined with respect to the bias of the assay results got. When the process is applied to the analyte in the presence of assumed levels of different components, compared the results got. When 27

the process is applied to the analyte in the presence of assumed levels of other components, compared to the results got on the same analyte without added substances. 1.7.7 Robustness and Ruggedness: The robustness is the analytical capability, when measured on deliberate differences in method parameters, final values remains unaffected by these variations in parameters; it is reliability shown during normal usage. Robustness of an analytical method was identified by changing the ph of the buffer and found to acceptable for organic addition, the 5 % v/v change in organic was influencing the retention time of peaks, hence method validation advice to be cautious while adding organic phase. The ruggedness is degree of redevelopment of results, when measured under a various of Laboratory conditions, other analyst, column and environmental situations, but followed specified analytical parameters given in method, final values remains unaffected by these variety of conditions; it is reliability indication during normal usage, this is recommended when method is to be changed to be used in more than one lab. 1.7.8 Stability of Solutions: Stability of a standard solution, sample solution and reagents used for colour development reaction is necessary for a reasonable time to 28

generate reproducible and accurate results. For example, 24 h stability is sufficient for all solutions and reagents that necessary to be prepared for each analysis. 1.7.9 System Suitability: Test assurance of system suitability, accuracy and precision of results on a specific occasion, system suitability tests performed every time while method is used either before or during analysis. The results of system suitability test are compared with standard values criteria, the method are satisfactory on that occasion if values are within limit of creiteria. A 1.8 Stastistics-treatment of Analytical Data [132-134]: Collecting, organising, summarising and analysing data from experiments are important elements in the presentation of all scientific data. Laboratories produce data that are associated with some degree of variability that affects the evaluation and interpretation of the data, therefore knowledge on degree of uncertainty is essential. Statistics forms the basis of this knowledge and is the key to drawing valid conclusions and making reasonable decisions. Statistics is defined as a science of data collection, interpretation of numerical data, presentation and analysis. Statistics is also the key to condensing data into a form that makes main features of a data clearer. The term error is defined as the 29

departure of the computed value from its real value. Thus, most of the difference between the two values, greater will be the magnitude of error involved. A large proportion of observations in a group of observations on some variables have a tendency to cluster around some central value. This is known as central tendency. Mean is calculated by adding all data values and dividing added value by the number of measurements, X= X/n. Median is one of the measures of central tendency. It divides the ordered sequences of data into two equal groups, half of the values will be less than the median and half will have values greater than the median. If n (number of observations) is odd there will be one and only one middle value or median i.e. ½ (n+1) th observation from both ends of the order of sequence. If n is even there is no middle observation but the median is declared by convention as the mean of two middle identifications, i.e. the (n/ 2) th and ((n+1)/2) th observation from one end in the ordered sequence [70]. The difference between the observed value and arithmetic mean of the set of observations in known as deviation, di = Xi X. Mean deviation is the arithmetic mean of all deviations irrespective of all the sign of the d d2 d3 deviation d = n 1 dn The standard deviation of an infinite set of data is theoretically the mean of the squares of the difference between the individual value xi and 30

the mean of the infinite observation µ. Scientific experiments often have a small number of observations. Standard deviation is calculated using (n- 1) observations because it represents better estimate of the standard deviation of a small population from which the sample is taken, S = (x ) 2 i n 1 For large number of observations (n-1) = n. Therefore, S = (x ) 2 i n The variance of a set of data is a square of the standard deviation. Variance (standard deviation) measures the extent to which the data vary amongst themselves. The relative measures of dispersion is expressed by the term coefficient of variance for a set to measurements having X (Mean) and S (standard deviation), the coefficient of variance can be computed as S 100 X The statistical interval set about the mean X, so that, for a given number of replicate measurements and for a given probability level the population mean µ lies within the specified range. These statistical limits are known as confidence limits and the interval so generated is called as a confidence interval. Confidence interval = X S. D. N X z 31

Where, X is the observed mean, SD is standard deviation for the experiment containing N number of observations. z is the student t value obtained from the 12 student t table for the given level of confidence and for a given degree of freedom. Confidence intervals are usually calculated at 95 % and 99 % level of confidence. The smaller the confidence interval at 95 % and 99% levels of confidence, the more precise are the results. The statistical tool to expect the unknown values of one differenciative from known values of another differnciative is called as regression. In regression analysis the average relationship between the two variables is revealed and thus it is possible to make a prediction of the dependent variable. The variable whose value is influenced is called as the dependent variable Y ; the variable, which exerts the influence, is called as the independent variable X. In case of linear regression, it will be seen that a unit change in the value of the independent variable (X) will produce a constant and absolute change in the dependent variable (Y). When the two variables have a linear relationship the regression line can be used to determine the value of the dependent variable. The correlation coefficient obtained during the analysis of a regression line plays an important role in deciding the relationship between the independent and the dependent variable. Most cases analytical methods are based on a calibration curve in which a calculated quantity y is plotted as a function of the known 32

concentration x of a series of standards. A plot of obtained data could be a straight line. However, the complete data points fall exactly on the line because of the random errors in the calculation process. Thus, we try to derive the best straight line from the points. A statistical technique called the method of least squares provides the means for objectively obtaining an equation for such a line and also for specifying the uncertainties associated with its subsequent use. In applying the method of least squares, one has to assume, that there is a linear relationship between the peak area (y) and the analyte concentration (x), by the equation y = mx + b, in this, b is the intercept (the value of y when x is zero) and m is the slant of the line. Residual value is the vertical difference of each point from the straight line. Total of the squares of the residuals minimizes by the line generated by the least-squares method. 33