Introduction to Impurity Profile

Transcription

1 Chapter 1 Introduction to Impurity Profile 1.1. INTRODUCTION Pharmaceutical drug stated as medicine or medication, officially termed as a medicinal product. Pharmaceutical derives from the Greek pharmakeutikos. A pharmaceutical drug is any chemical substance which may be used or administrated to diagnose, treat, cure or to prevent the disease or the other abnormal conditions ( 2010). As per regulatory guidelines, pharmaceutical drug defined as it is any chemical substance that is recognized by any regulatory body or official pharmacopoeia proposed to diagnose, treat, cure or to prevent the disease or the other abnormal conditions in man or animals ( US Federal Food, Drug, and Cosmetic Act, 1938). For many centuries, human civilization has been developing and consuming drugs, but it is only in last century that enormous and systematic research has been done on development of pharmaceutical drugs. The average life span of human is increased by the use of drugs by curing and preventing diseases. These drugs are manufactured in bulk and formulated into convenient dosage forms for their desired therapeutic use. These formulations should be stable, nontoxic and in acceptable state, confirming safety, quality and efficacy. (Gorog, 2000; Ahuja, 1998; Singh et al., 2012). Safety, quality and efficacy of drug substances are the fundamental concerns in drug therapy. Safety and quality of pharmaceutical substance is monitored by its pharmacological profile and/or toxicological profile and the adverse effects caused by its impurities. The drug should be safe, i.e. it should have acceptably low risk of adverse effects with doses of drug which provide the wanted therapeutic effects. Thus quality of drug is directly related to safety. The quality as well as safety of a drug is guaranteed by screening it using suitable analytical techniques. Therefore, the analytical techniques and related events about drug impurities are most main subject in pharmaceutical analysis (Ahuja, 2007, Smith and Webb, 2007). 1

2 Until 1990s, there was no precise definition for impurity in the pharmaceutical world, because of the apparent negativity attached to this word. The definition for impurity in Webster s dictionary is something that is impure or makes something else impure. In the pharmaceutical industry, it is the latter meaning that is frequently attached to the meaning of an impurity. A simple definition of impurity has been offered: impurity is a material which disturbs the purity of the material of target, viz., drug substance or drug product IMPURITY AND ITS DEFINITIONS IMPURITY An impurity is any constituent present in excipient, drug substance (Active Pharmaceutical Ingredient or bulk material) or drug product (Dosage form or Finished product) that is not the an excipient, active drug substance, formulated drug product. This definition of impurity is broad enough to include degradation products as impurities. The term degradation product (DP) is defined in ICH as follows: ( ICH guidelines, 2012) DEGRADATION PRODUCT (DP) A molecule resultant from a modification in the active drug substance or formulated drug product brought over time SOURCE OF IMPURITIES Bulk drug Substance process development and formulated drug Product formulation development are two main areas of the pharmaceutical drug development process. Impurities can be produced in either of the processes. During the Drug Substance synthesis development process, impurities can be generated from the synthetic process or as a result of degradation. In drug product formulation development, impurities can also be generated as the degradation products, as a product of drug excipient interaction, or external contamination or from packaging components CLASSIFICATION OF IMPURITIES Theoretically possible (potential) impurities are classified (Figure 1.1) as following types (Guidance for industry, 1998). 2

3 Figure 1.1: Classification of theoretically possible impurities PRIMARY IMPURITIES These impurities are generated along with development of a wanted product, like byproducts, deposits of starting ingredients and intermediates SECONDARY IMPURITIES These impurities are deposits of impurities of starting ingredients, degradants of primary impurities, yields of reactions among impurities etc NON GENOTAOXIC IMPURITIES Non genotoxic impurities are ordinary impurities those don t have any harmful effect on genetic material GENOTOXIC IMPURITIES Genotoxic impurities harm the organism by damaging its genetic material (DNA). Based on the source of impurities they are classified (Figure 1.2) as follows Figure 1.2: Classification of Impurities based on their source 3

4 1.5. DESIGNATION OF IMPUIRTIES Impurities have been titled differently by various groups of scientists who deal with them. Commonly used terms are displayed in Figure 1.3 and those terms have been found acceptable by ICH and various regulatory bodies. Figure 1.3: Designation of Impurities 1.6 IMPURITIES AND REGULATORY GUIDANCE Ethical, economic and competitive reasons as well safety and efficacy reasons it is important to monitor impurities in drug products (Mollica et al., 1978) However, monitoring and controlling these impurities mean different to different individuals or to the same individuals at different times, even those in pharmaceutical sciences and industry. A unified terminology is needed to assure that everyone uses the same vocabulary when addressing questions related to impurities. In this context, the leadership delivered by ICH is very helpful. USFDA have recommended the guidance made under ICH to improve the quality, safety and efficacy of drug therapy. The guidance, developed with the combined efforts of regulators as well as industry legislatures from the European 4

5 Union, Japan and the United States, has aided to confirm requirements for the data that should be produced to different regulatory organizations. Summary of the current Guidance that pertains to impurity information for NDA and ANDA products is tabulated below in Table IMPURITIES IN DRUG SUBSTANCES Purity of a drug substance depends on lowering the level of impurities, not only at the time of release, but also maintaining low levels of degradants during the shelf life of the drug substance. Procedures are usually proposed to monitor and control synthetic precursors, synthesis-related materials, intermediates, heavy metals, moisture and volatile solvents. Stability indicating methodology that can differentiate between drug substance and degradation impurities, process impurities or other potential degradants is necessary to monitor the degradation profile over a long period of time. Starting the synthesis with high purity materials certainly adds to the purity profile of the final product. The impurity profile in a new drug substance (ICH guidelines, Q3A, 2006) may change for a variety of reasons, such as synthetic route changes, process scale-up changes, and changes made to key starting materials and intermediates. ICH decision trees help classify, qualify, and select limits for new molecular entities (NMEs).Thresholds for the impurities in bulk substances are listed in Table 1.2. In many cases, studies executed to qualify an impurity or degradant will depend not only on the maximum daily dose intake, but also on the population of patients, route of administration and duration of drug administration IMPURITIES IN FORMULATED DRUG PRODUCTS ICH Guidance includes the degradation product(s) of bulk drug substances (New Molecular Entities, NMEs) in the formulated product and process products of the bulk drug substance. Below Table gives a summary of limits for impurities in formulated drug products, from the ICH Guidance. 5

6 Table 1.1: Regulatory guidance on Impurities 6

7 Table 1.2: Limits for DPs in drug substances During NDA filing, the proposed limits should address Every specified DP Any unspecified DP Total DPs ICH has given threshold limits for qualification, reporting and identification of degradants in formulated products as follows (Table 1.3) (ICH guidelines, Q3B, 2006) GENOTOXIC IMPURITIES Genotoxic impurities harm an organism by changing or damaging its genetic material (DNA). However there exists a safety document amendment of the ICH S2 Guidelines dated March 2008: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (S2A) and Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals (S2B). These guidelines are mainly for testing of pharmaceuticals for genetic toxicity. 7

8 Table 1.3: Limits for DPs in formulated products The official FDA draft guideline entitled Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approaches was published in December 2008 ( FDA guidelines, 2008). The intention of the draft guideline is to be an adjunct to the guidelines of ICH Q3A, B and C. Recommendations from the CHMP document are acceptable for exposure threshold limits for clinical development and marketing applications. FDA recommended approaches for initial assessment of genotoxic potential is similar to that mentioned in 8

9 the literature above. The recommended approach for handling genotoxic and carcinogenic impurities is either prevention or reduction of those impurities IMPURITY PROFILING The name impurity profiling is commonly reflected to be a set of analytical activities targeting at the detection, identification or structure characterisation and quantitative estimation of organic, inorganic impurities, and residual solvents, in drug substances as well as in a drug products. As per ICH guidelines an impurity defined as Any component of the medicinal product which is not the chemical entity defined as the active substance or an excipient in the product, while impurity profiling is considered A description of the identified and unidentified impurities present in a medicinal product ( ICH guidelines, 2012). Table 1.4: Classification of genotoxic potential impurities as per PhRMA SIGNIFICANCE OF IMPURITY PROFILING IN DRUG SAFETY Regulation impurities in API and formulated products were controlled by several regulatory agencies like US-FDA, EMEA, ICH, TGA and MHRA, etc. Recent 9

10 days apart from drug purity profile there was an increasing importance of impurity profile by regulatory authorities. Hence reporting, identification and qualification of impurities are required for determining the biological safety of each impurity IMPURITY PROFILING IN SYNTHETIC DRUG RESEARCH In the field of synthetic research, compounds are generally synthesized in lab scale or small scale and only after the preliminary screening for drug activity the role of impurity profiling begins for certain molecules which have shown desired activity. Organic chemist has to optimize the synthesis and purification steps of the drug substances to prevent or reduce the occurrence of impurities so that these can be scaled up to prepare drug for formulation and for toxicological, preclinical and clinical trials. It is important to identify the impurities at this stage of development (Gorog, 2003) IMPURITY PROFILING IN PRODUCTION OF BULK DRUGS After the introduction of new API, it has to be synthesized in bulk level for formulation. It is very important that there should not be any new impurities appear during the scale up procedure and quantity of impurities in bulk drug material identified during the R& D phase remains below specification limit IMPURITY PROFILING IN FORMULATION RESEARCH AND DEVELOPMENT (FR&D) FR&D people should have the knowledge of impurity profiling of bulk drug used for development of formulation, which helps them in discriminating synthesis related impurities and degradation products. Stability studies are carried out to classify the degradation products from synthesis related impurities. The amount of degradation products increases over time while that of synthesis related impurities remain unchanged. These studies also help the formulation scientist to initiate the preformulation studies IMPURITY PROFILING IN DRUG REGISTRATION The manufacturers of the pharmaceutical drug substances have to submit a drug master file (DMF) to the regulatory authorities. The document provides 10

11 information about the facilities, process used in the manufacture, packaging and storage conditions of the drug in detail. It also contains impurity profile along with their acceptance criteria. In the area of impurities, through the efforts of ICH and US- FDA a tremendous amount of information is available. The comparison of impurities in different batches of same product from same manufacturer provides us a good sign for consistency in the manufacturing process while comparison with other manufacture of same drug indicates us to the differences in the purity and impurity profile. The impurity profiling also indicates the synthetic route of different companies as some impurities are specific to the synthetic pathways (Guidance for industry ANDA, 1998). Therefore it is very important to characterize each impurity generated during synthetic process, during storage or during formulation due to drug-excipient interaction. After elucidating the structure we will able to find the cause of formation of the impurity and can control it to a low limit that will not affect purity, safety and potency of formulated product. Thus the importance of impurity profiling can be summarized as follows 1. By identifying the structures and finding the route of formation of impurities the organic chemist can change the synthetic process or reaction conditions to avoid or to decrease the impurity to an acceptable limit. 2. The characterized impurity further synthesized or isolated, used as an impurity standard and used in quality control testing of every batch. 3. These impurities can be targeted to toxicological studies to prove the biological safety of the drug IMPURITY PROFILE AND STABILITY STUDY Stability study Formal stability study Stress testing/forced degradation study Physical Chemical Microbial stability stability stability Long term stability study Intermediate stability study Accelerated stability study 11

12 Stability as well as stressed degradation studies are essential part of impurity profile, which provides the intrinsic stability of API or drug product and formation of degradation products under different stressed conditions. ICH guidelines for stability testing are out lined in Table 1.5. Table 1.5: ICH guidelines for stability study ICH Code Q1A Q1B Q1C Q1D Q1E Q1F Zones Q5C Guideline title Stability testing of New Drug Substances and Products (Second Revision) Stability testing: Photo stability testing of New Drug Substances and Products Stability testing of New Dosage Forms Bracketing and Matrixing Designs for stability testing of Drug Substances and Products Evaluation of stability data Stability data package for Registration Applications in Climatic III and IV Stability testing of Biotechnological/Biological Products FORMAL STABILITY STUDY The intention of storage or stability testing is to deliver confirmation on how the quality of active drug or formulated product differs with time under the influence of light, temperature and humidity (Reynolds et al., 2002; ICH guidelines, Q1A, 2003). In principle, the influence of each factor is first individually explored, then cross-influences are evaluated. The duration of the study will depend on the studied factor and the sensitivity of the product to that factor. Stability study conditions are varied with climatic zones shown in Table 1.6. Based on the duration of time and conditions employed, stability study is categorised as displayed in Table 1.7and Table

13 Table 1.6: Climatic zones for stability study (ICH guidelines, Q1A) Climatic Zone Climate/definition Major countries/region Zone I Temperate United Kingdom, Northern Europe, Russia, United States II Subtropical and Mediterranean Japan, Southern Europe III Hot and Dry Iraq, India IVa IVb Hot and humid Hot and very Humid Iran, Egypt Brazil, Singapore Table 1.7: Stability testing conditions for zone I and II (ICH guidelines, Q1A) Table 1.8: Stability testing conditions for zone III and IV (ICH guidelines, Q1A) INFLUENCE OF TEMPERATURE AND HUMIDITY Temperature has great influence on all range of reactions and generally they are accelerated by increase in temperature. The behaviour of the drug substance under extreme temperatures such as 50 C or even 70 C should be investigated. The ICH guidelines on stability, recommend a 10 C increment above the accelerated conditions (40 C).Also sensitivity of drug substance towards relative humidity (RH) from a dry atmosphere up to a water-saturated atmosphere (from 10 to 90% RH) was investigated. Temperature and humidity will influence the physical stability. 13

14 INFLUENCE OF ph Acidic and basic ph has impact on the degradation of most of drugs. Rise or decrease in ph may degrade the active drug or formulated product. The outcomes of these studies will serve to explain or to better select some conditions for active drug substance and formulated drug product manufacture, along with for preformulation studies INFLUENCE OF OXYGEN Oxidation is the significant pathway of drug degradation. Oxygen will decompose drug substance through auto oxidation. Oxidative degradation of drugs can be separated into types: react with molecular oxygen and react with other formulation oxidizing agents. Oxidation in tablet formulation dosage form depends on hardness or presence of coating which affect the oxygen permeation rate STRESS TESTING (FORCED DEGRADATION STUDY) Stress degradation study often termed as forced degradation study can be differentiated by the focus of study and the severity of conditions used from that of accelerated stability study. As this study is an examination of intrinsic strength of the molecule, provides the information for analytical method development and validation. Stress stability studies are intended to evaluate stability problems, which will affect different areas as follows Analytical method development Development of Formulation and package Suitable storage & shelf-life establishment Manufacturing factors 14

15 Table 1.9: General procedure for degradation study of drug substance and drug product 1.8. IMPURITY PROFILE AND ANALYTICAL METHODOLOGY With the high quality standards required of pharmaceutical products, there is a great need to separate, quantify, isolate and determine the structure of impurities present in the final active drug and formulated product. These impurities are present at extremely low levels or trace level and analytical characterization could by itself be a technical challenge.thus there is abundant need for establishment of analysis methods for quality of new drugs. Pharmaceutical analysis (Beckett and Stenlake, 2004) deals not only with medicaments (drugs and their formulations), but also with their precursors i.e. with the starting material on which degree of purity and the quality of medicament depend. The quality of a drug is determined after establishing its purity and the quality in the drug substance and its formulations. Quality (Sethi, 1997) is more significant in every product or service but vital in medicine hence involves life of human or animal. Unlike ordinary consumer goods there can be no second quality in pharmaceutical drugs. The quality of analysis is a crucial feature in the accomplishment of drug development. Analytical test method is a specific procedure by application of a technique to carry out the analysis in order to solve an analytical problem. Analytical field instrumentation shows a substantial role in the estimation and production of new products and affords the lower detection limits essential to assure safety of food, drug, water and air. Physicochemical methods are used to study the physical phenomena that occur as a result of chemical reactions. In instrumental analysis physical property of a material or substance is measured to decide its chemical configuration. Often it is 15

16 necessary to use more than one instrumental technique to obtain the information required to solve the analytical problem. Instrumental methods used to save time or to obtain increased accuracy. The time saving factor should be considered where a considerable number of determinations are to be made in routine analysis. Most important and three principal areas in analytical methodology for pharmaceutical analysis are: Chromatography Spectroscopy Electrochemistry The analytical method plays a vital role in the dossier submission and it becomes inevitable to comply with the regulatory requirements. Regulatory authority states that the analytical method should be stability indicating which is employed for the analysis of assay as well as related substances of bulk drug and formulated drug product. The analytical method of related substances should be sensitive to estimate the synthetic or degraded impurities. The analytical method for the related substances paves the way for the impurity profiling, elucidation of degradation pathway, mass balance etc. Different dosage forms with various excipients pose challenge during the development of analytical methods. Standard analytical procedures for the estimation of assay, related substances, dissolution may not be existing in Pharmacopoeia for all the products; hence it becomes essential to develop new analytical testing methods in such a situation. The estimation of degraded and synthetic impurities in the presence of the analytes can be analysed by HPLC, GC, HPTLC, UPLC, LC-MS, LC-MS/MS, GC-MS, CE techniques. A general strategy can be set for the detection, separation, quantification, isolation, identification /characterization of the impurity of active drug and formulated product by the use of analytical techniques. The schematic use of the analytical techniques for impurity profiling of drug substances is shown in Figure 1.4. The impurity profile includes the identification of the key impurities in the intermediates, determination of residual solvents and inorganic impurities in the drug substances. As it mentioned above, the most important three principal areas of analytical methodology are: Chromatography Spectroscopy Electrochemistry 16

17 Since, Chromatography and Spectroscopy techniques have been used in present research work, these techniques discussed in detail as below CHROMATOGRAPHIC TECHNIQUES Among all the different types of analytical methods, chromatography has unique application to resolve all types of analytical problems. Chromatography is an analytical methodology widely used for the separation, identification, and quantification of the chemical components in complex mixtures such as pharmaceutical formulations. No other separation method is as powerful and generally applicable as is chromatography. In the pharmaceutical industry, high performance liquid chromatography (HPLC) is the key and integral analytical device useful in all stages of drug discovery and development as well as in production. Chromatography was developed by the botanist of Russia, Mikhail S. Tswett (1903) (Tswett and Protok, 1930) and from then huge development was materialized of this technique. Tswett devised the name chromatography (chroma=color, and graph=writingliterally, color writing) to define this colourful experimentation. Since then there has been an huge progress of chromatography technique. Today, this chromatography, in its different methods, has become one of the most dominant tools in the analytical chemistry. Chromatography may be analytical or preparative depends upon its application and scale of loading. Analytical chromatography mainly used for separation and quantification of mixture of analytes whereas preparative chromatography used for separation followed by collection of analytes in order to get pure individual compounds. There different types of separation and quantification techniques widely available and used in impurity profiling are described below SEPARATION AND QUANTIFICATION TECHNIQUES Detection of known or unknown degradant/impurity is the major phase in impurity profiling. The estimation of organic impurities of the three types of impurities is the most interesting and challenging task. Following are the different techniques used for the separation as well as quantitation of impurities and DPs. 17

18 Figure 1.4: Analytical flow in Impurity Profile 18

19 HPLC, High Performance Thin Layer Chromatography (HPTLC), Gas Chromatography (GC), Thin Layer Chromatography (TLC), Capillary Electrophoresis (CE), Supercritical Fluid Chromatography (SFC), and Gel Permeation Chromatography (GPC).Recently Ultra Performance Liquid Chromatography (UPLC) is emerging as a fast separation liquid chromatographic technique. Since, we have used only HPLC and UPLC in present work, these techniques are described in detail as below HPLC The chromatographic methods are characterized by high sensitivity, selectivity and economical consumptions of chemicals. HPLC is a chromatographic tool used for separation and quantification analysis of mixtures of chemical compounds. HPLC can be operated in both modes i.e. reverse phase and normal phase mode. Reverse phase analysis involves use of polar mobile phase (e.g. water, methanol, acetonitrile, etc.) along with non-polar stationary phase like C8, C18, phenyl etc. Normal phase analysis involves use of non-polar solvents (e.g., hexane, dichloromethane, ethyl acetate etc.) along with polar silica as a stationary phase. Reverse phase analysis is useful for polar compounds (e.g., amines alcohols, acids etc.) while normal phase provides separation of non-polar compounds. Basic Instrumentation of HPLC showed in Figure 1.5 Figure 1.5 Basic Instrumentation of HPLC 19

20 A typical HPLC instrument consists of following components a. Mobile phase reservoirs b. Pump c. Injector d. Column & column oven e. Detector f. Control & data processing system g. Waste Mobile phase containers: These are inert mobile phase storage units. Pump: pump force mobile phase through the HPLC at a precise flow. Typical pumps operates at psi (400to 600bar) pressures. Pump can deliver in isocratic or gradient passion. A binary system will have two pumps and two channels to deliver two solvents either in isocratic or gradient mode. Thus the gradient mixing is shaped under high pressure. Hence these are called High pressure gradient systems (Figure 1.6). A quaternary system will have one pump and four channels to deliver four solvents. In this kind of systems a gradient proportioning valve (GPC) is ahead of the single pump; thus the gradient mixing is shaped under low pressure. Hence these are called Low pressure gradient systems (Figure 1.7). Figure 1.6 High pressure gradient instrument 20

21 Figure 1.7 Low-pressure gradient instrument Injector: Injector introduces the sample. Column and column oven : Column considered as the heart of the chromatograph, its stationary phase separates the components of a sample. Column oven maintain the temperature of column at a defined set values. Some of the column parameters are listed below. Container Length of column Diameter of column Material Packing materials Packing material Particle size Pore size Shape of packing material Surface area Carbon content Chemistry of bonding Silanol End-capping Purity Different column dimensions and a number of column chemistries are available which are very frequently used in analytical HPLC, Preparative HPLC and process scale level. 21

22 Detector: Detector can detect the different molecules elute from the column based on their physical and chemical characteristics. A detector measures the amount of constituents as per their concentration, useful for quantitative analysis of sample constituents. There are different detection principles employed to detect the mixtures eluting through a LC column based on the chromatographic properties of compounds and type of analytical application. The most common are: Spectroscopic Detection (UV/Visible, PDA and MS detectors) Refractive Index Detection (RI) Fluorescence Detection Other detectors are: Conductivity detector Electrochemical detector (ECD) Evaporative light scattering detector (ELSD) Charged aerosol detector (CAD) Control and data processing system: Often called as data processing system, the computer control all the units of the instrument as well as receipts the signal from the detector used to define retention time (qualitative- analysis) and the concentration of sample (quantitative- analysis). Data shall be processed after completing the analysis and in general retention time, peak height, peak area parameters are used for qualitative and quantitative analysis (Figure 1.8). Waste: The liquid mobile phase comes out from the detector can be sent to waste HPLC SCALES OF OPERATION HPLC can be categorised in to three types by its scale of application. a. Analytical scale-for qualitative and quantitative analysis b. Preparative scale-for isolation and purification analysis c. Process scale-for higher scale purification analysis 22

23 Figure 1.8: Acquisition of data Detector output Scaling Amplifier Analogue to Digital converter Interface Computer Presentation of Results ADVANTAGES OF HPLC High resolution capacity Superior qualitative and quantitative ability and reproducibility Reasonable analytical settings Sample does not need to be vaporized like GC High sensitivity Less sample utilisation Easy prep LC separation & purification APPLICATIONS OF HPLC Analysis of Biogenic substances Analysis of Medicinal products Analysis of Food related products Analysis of Environmental related samples Analysis of industrial products 23

24 ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC) Advancement of HPLC is continuously encouraged to improve the efficiency of any one or more aspects of chromatographic analysis. UPLC utilises improvement of technological advances made in system, detector design, particle chemistry, and data processing and control. UPLC improves the chromatographic analysis in three aspects, namely, chromatographic resolution, speed and sensitivity in analysis. In UPLC, a column composed of sub-micron particles, a pump with higher pressure and a detector with higher sensitivity than they are used in HPLC. Therefore UPLC analysis protects time and decreases solvent usage (Swartz, 2004; Jerkovich et al., 2003; Wu et al., 2001). As size of particles reduces to below 2.5μm, there is a important improvement in efficiency and it doesn t reduce at increased linear velocities or flow rates as per the common Van Deemter equation UPLC SYSTEM A typical UPLC system (Waters ACQUITY TM ) is depicted in Figure Figure 1.9: Waters ACQUITY TM UPLC system The Van Deemter curve [Figure 1.10], ruled by an equation with three components (Wu et al., 2001; MacNair et al., 1997) 24

25 Figure 1.10: Van Deemter plots-influence of particle size H= A + B/v + C v Where A, B and C are constants ν is the linear velocity. The evolution of particle size chemistry illustrated in Table Table 1.10: Development in Particle technology for Liquid Chromatography (Majors et al., 2005) Year(s) of Acceptance Most Popular particle Size ~ Plates/15 cm 1950 s 100 μm μm μm μm μm μm (pellicular) μm ( poroshell) μm μm

26 Sample injection: UPLC works at higher pressure sample introduction is critical. To guard the UPLC column from extreme pressure fluctuations, pulse-free injection procedure is introduced and the swept volume of the UPLC is minimised to decrease potential band spreading. UPLC columns: Van Deemter equation applications fulfilled with smaller particles. Resolution is increased in a sub 2 micron particle packed column. Separation of the constituents needs a bonded phase that offers both retention and selectivity. The design and development of submicron column particles is a major challenge (Jerkovich et al., 2003; Wu et al., 2001) ADVANTAGES Reduces run time & improves sensitivity Offers wider dynamic range & selectivity fast resolving power of UPLC Reduced operation cost Very less consumption of solvent More productivity existing resources DISADVANTAGES Reduced the life of the columns with high back pressure METHOD DEVELOPMENT Detection, Identification and quantification of related impurities is fundamental task in drug process development for the evaluation quality and safety. Presence of impurities in pharmaceuticals even in trace levels may impact the safety, efficacy and quality of the pharmaceutical drug products. Therefore there is great requirement for the development of new analysis methods for safety and quality assessment of new drugs. Analytical method development is a scientific skill / art to develop suitable methods based on strong scientific background of samples to be analyzed and instruments to be used for analysis. Analytical methods for impurities estimation / impurity profiling should be stability indicating to monitor the stability of pharmaceutical dosage forms. Development of any new or improved analytical method for the analysis of an analyte usually depends on tailoring the existing analytical approaches and 26

27 instrumentation. Method development (Yord et al., 1997; Ahuja and Dong, 2005) usually involves choosing the method requirements and type of different instrumentation. In the development phase, decisions of choice of mobile phase, detectors, column, and methods of quantification must be addressed. In this way, development considers all the parameters pertaining to any method. There are valid reasons for new method development: a) The drug or combination of drug may not be as official in any pharmacopoeias b) There may not be a suitable method c) Existing methods may not be accurate, contamination prone, or they may not be reliable d) Existing methods may be much expensive, time taking, or they may be easily not automated. e) Existing method not sufficient sensitivity f) Newer instruments and techniques may have developed that offer opportunities for upgraded method Development of method can be established on sample nature and different goals and existing resources but some basics can be discussed. The major and important steps involved in analytical method development are briefly discussed below. Literature search Selection of type of chromatography Stationary phase selection Mobile phase selection Detector and wavelength selection Diluent selection Selection of separation goals Optimization of separation Optimization of test concentration and injection volume LITERATURE SEARCH Any development work starts with literature survey of existing methods and useful information about compound. The purpose of literature search is to collect all the information available in public domain like from journals, abstracts, pharmacopoeias (USP, EP, JP, IP), patents and innovator summary basis of approval (SBOA).Thorough literature search enables to get information as follows 27

28 Solubility profile of drug: Solubility data of drug in different solvents and at different ph conditions useful in diluents selection and mobile phase selection. Analytical profile of drug: Physico chemical properties of drug like polarity, pka, melting point, degradation pathways will help in selection of ph of buffer, organic modifier and storage conditions of drug. Major functional groups and their polarities are shown in Table Stability profile: Stability data of drug gives the information about of sensitivity of drug towards ph, light, heat, moisture etc. Sample, impurities and degradation products: Information about sample, impurities and degradation products and their polarities, structures, major functional groups will help in selection of mobile phase and stationary phase SELECTION OF MODE OF CHROMATOGRAPHY Selection of mode of chromatography is crucial and primary step in method development. Reverse phase chromatography is ideal choice of majority of pharmaceutical compounds. Table1.12 illustrates the selection of type of chromatography based on the sample nature. Table 1.11.Major functional groups and their polarities 28

29 SELECTION OF STATIONARY PHASE The stationary phase is the heart of the chromatograph and the failure or success of the analysis is depending on the operating conditions of the columns. The variables in the column packing are the bonded phase, end capping, pore size, carbon load and surface area. The variables in the column configuration are the length, internal diameter and material of construction (stainless steel, plastic). Selection of stationary phase is purely based on the polarity of molecules to be analysed. Columns with silica different cross linkings in increasing order of polarities as follows: Non-polar moderately polar polar C-18 < C-8 < C-6 < Phenyl < Amino < Cyano < Silica Each stationary bonded phase has unique selectivity for different sample categories. Table 1.13 illustrates commonly used bonding phases and their properties. Column Parameters and their specifications/ properties are described in Table 1.14and Table Table 1.12.Selection of mode of chromatography 29

30 Table 1.13.Types of bonding phases and their properties Common bonding phases Chemical structure Polarity Mode of chromato graphy Retention mechanism C-18 Very non polar Reverse phase London/dispersion/van der waals interactions with hydrophobic compounds C-8 Non polar Reverse phase London/dispersion/van der waals interactions with hydrophobic compounds Phenyl Non polar Reverse phase Mixed mechanism of hydrophobic and π- π interactions Cyano Intermed iate polar Reverse and normal phases Mixed mechanism of hydrophobic, dipoledipole and π- π interactions Amino Polar Normal phase and ion exchange Dipole dipole interactions or acid base interactions Unbonde d silica Very polar Normal phase Hydrogen bonding Column Parameters Dimensions of column (Table 1.14) Packing material properties (Table 1.15) Length of column Silica based packing material Diameter of column Polymer based packing material Particle size Particle shape 30

31 Pore size Surface area Carbon loading Chemistry of bonding End capping Table 1.14.Column dimensions and their effects on chromatography Common column name Column length in mm Column diameter in mm Effect on chromatography Short column Short run times and low back pressure Long column Higher resolution and longer run times Narrow bore column < 2.1 Higher detection sensitivity Wide bore column Higher loading of samples SELECTION OF MOBILE PHASE Selection of mobile phases is purely depend on pka and polarity of molecules to be analysed and mode of chromatography chosen for analysis. Selection of mobile phase includes following parameters. ph of buffer Buffer selection Type of salt Buffer concentration Selection of organic modifier Solvent strength Ion pairing reagents ph of buffer: During separation of acids and bases in reverse phase chromatography, ph is needs to be controlled by proper buffer. For molecules which does not contain any ionic functional group ph control is not required. Selection of buffer ph depends on the pka of the functional group (Table 1.16) present in the molecule When analyte is ionized the hydrophobicity decreases and the retention will decrease. 31

32 Table1.15.Packing material properties and their effects on chromatography Packing material property Schematic representation Effect on chromatography Silica based material Higher mechanical strength and higher efficiency, acidic silanol effect on basic compounds. Polymer based material Longer life time, wider ph range, lower back pressure, lower mechanical strength. Particle size Smaller particles offer higher efficiency also high back pressure. Analytical column: 3-5 Semi preparative: 5-10 Preparative: Particle shape Spherical particles offer higher efficiency, longer column stability and lower back pressure. Pore size Surface area Carbon loading Chemistry of bonding End capping Pore size of 150A or less for sample MW Pore size of 300A or greater for sample MW > High surface area provides greater retention, capacity and resolution. Low surface area equilibrate quickly, especially useful in gradient analyses. High carbon loads offer greater resolution and longer run times for hydrophobic samples, Low carbon loads shorten run times and often show different selectivity.increase in carbon increases the retention. Normal range of carbon content is 12-20%. Monomeric bonding offers increased mass transfer rates, higher column efficiency, and faster column equilibration. Polymeric bonding offers increased column stability with pure aqueous mobile phases and higher sample loading. End capping reduces peak tailing of polar solutes. 32

33 Table 1.16.pKa values of functional groups Rule of thumb for selection of ph is as follows Acidic compounds: Better to use acidic ph mobile phase so that compound will be in unionized state and will retain more. Basic compounds: These compounds will be ionized completely and will elute early in acidic mobile phase; peak shapes are also better. These compounds will be unionized in basic ph mobile phase and will retain more but peaks may show tailing because of active silanols at basic ph. Neutral compounds: Neutral mobile phase is suitable. In reverse phase mode HPLC the retention of analytes is based on their Hydrophobicity. More hydrophobic leads longer retention As ph increases acids loss proton and ionized. Then it becomes less hydrophobic and more hydrophilic causing in reduced retention. As ph decreases bases gain proton and ionized. Then becomes less hydrophobic and more hydrophilic resultant in reduced retention The retention of acids, bases and neutrals against ph displayed in Figure

34 Figure.1.11: Retaining of acids, bases and neutrals against ph ph of mobile phase chosen ± 1.5 ph units away from pka of analyte. This keeps analytes are either 100% ionized or 100% non-ionized and should help to control run to run reproducibility. When mobile phase ph is close to the pka value of the analyte, even a minor change in ph will have major effect on the resolution. Dissociation of acids and bases at different ph values presented in Figure 1.12 and Figure Figure1.12: Dissociation of acids at different ph values Figure1.13: Dissociation of bases at different ph values 34

35 Buffer selection: Buffer provides continuous ionic strength to mobile phase. So, it is always better to employ buffer in aqueous state of the mobile phase for RP mode chromatography. Buffering in mobile phase increases the ruggedness of the test method. Select buffer strength of about 10 to 25 mm for initial experiments. The type of buffer to be s elected is purely depends on the pka of molecule. Type of salt: Since every salt consists of a particular pka value and ph range, selection of type of salt completely based on the ph of mobile phase which in turn depends on the pka of sample selected for analysis. As rule of thumb, analysis should be within ±1 ph unit of the buffer pka for effective ph control. Commonly used buffers in reverse phase chromatography and their properties are showed in Table Buffer concentration: Buffer concentration in the range of 10 to 50 mm is suitable for most RP chromatography applications. Selection of organic modifiers: Acetonitrile (MeCN) as well as Methanol (MeOH) are the leading choice for organic modifier. MeCN is best due to low UV cutoff as well as Low viscosity. is and so selectivity of MeOH and MeCN significantly different since MeOH proton donor and MeCN is proton acceptor. IPA & THF are other alternates. However, THF mobile phases are not stable. Mobile phases with MeCN, to avoid pumping difficulties with 100% MeCN, make always about 5-10% aqueous portion. Order of polarity: Methanol > Acetonitrile > Ethanol > THF > Propanol Order of solvents strengths: Propanol > THF > Ethanol > Acetonitrile > methanol SELECTION OF DETECTOR AND WAVELENGTH Select the detector based on the presence or absence of chromospheres. Majority of pharmaceutical compounds exhibit UV spectra in the region of nm. Initial experiment can be done with lambda max of drug substance. Non chromophoric compounds can be analyzed with ELSD/RI/CAD/MS.If compound fluoresce, it can be analyzed by using FLD. Commonly used detectors are described in Table

36 Table 1.17.Commonly used buffers in reverse phase chromatography Buffer pka Buffer range UV cut off (nm) Phosphate pka pka pka Citrate Carbonate pka <200 pka Tris Borate Ammonium acetate pka pka Ammonium formate Trifluoro acetic acid 0.5 Acetic acid Formic acid Triethyl amine Ammonia Pyrrolidine SELECTION OF DILUENT Select the diluent for APIs in which the API is completely soluble and peak shape is good. Select the diluent for finished dosage forms, in which the analyte should be extracted from excipients. Calculate the % extraction against pure compound in the concentration of linear range, (preferably < 1 AU) by diluting the test. The peak shapes of all compounds should be symmetrical in the selected diluent. Select the diluent in such a way that the drug substance or drug product and its impurities, degradation products, intermediates should be soluble in the diluents. The diluents should be compatible with the mobile phase to have better peak shape of the analyte. 36

37 Table 1.18 Commonly used detectors and their selection SELECTION OF SEPARATION GOALS The goals and requirements desired to achieve during method development should be decided before starting the method development activity. 1. Base to base separation between all the impurities 2. Base to base separation between impurities and placebo peaks (if any) 3. Base to base separation between all the impurities and principal analyte peak. 4. base-to-base separation between placebo peak(s), if any and Principal analyte. 5. Good peak shapes for all the impurities. 6. Adequate Run time and simplicity in method OPTIMIZATION OF SEPARATION After getting the initial separations by setting the above parameters we need to optimize the method to achieve the desired separation goals by optimizing the method parameters as follows Changing the gradient programme 37

38 Changing the ph Introducing mobile phase additives Introducing different organic modifiers or ion-pair reagents in mobile phase Changing the column chemistry and its parameters OPTIMIZATION OF TEST CONCENTRATION AND INJECTION VOLUME After finalization of chromatographic conditions (gradient programme, mobile phase, column and Diluent) fix the test concentration to get the LOQ of all impurities and analyte less than the reporting threshold. In case the LOQ s are not meeting the requirement, increase either the test concentration or injection volume to get the desired LOQ or change the selective detector for specific compounds which can give higher response ANALYTICAL METHOD VALIDATION Analytical method validation is an integral part of product development. The developed analytical methods are validated in order to establish that it is appropriate for its intended purpose. Validation is defined as finding or testing the truth of something. Validation is essential for any method to confirm that it is accomplished to give reproducible and reliable results. The validation documents have to submit to the regulatory agency on a well-documented procedure. The ICH was provided guidelines for method validation (ICH guidelines, Q2B, 2005; ICH guidelines, Q2A, 2005). Typical parameters for analytical method validations are A) Precision B) Specificity C) Accuracy D) Limit of Quantitation E) Limit of Detection F) Linearity and Range G) Ruggedness H) Robustness 38

39 PRECISION The precision sectioned into three types; repeatability (Intra-day precision), intermediate precision (inter-day precision) and reproducibility (between laboratories). Repeatability is the sameness of test results obtained using same method on identical sample in same laboratory, by same operator and on the same equipment. Intermediate precision refers to the sameness of test results obtained from random changes such as different analysts, equipment, experimental period. Reproducibility is the sameness of test results obtained using same method on identical sample in different labs, by different analysts and on different instrument SPECIFICITY Specificity is ability to quantity the analyte accurately in the existence of all other sample materials. Specificity determination can be performed in two ways. 1. Obtaining resolution greater than 2.0 between all the known and unknown degradants. 2. Selection of right detector to get selectivity. Mass balance is superior quality control test on analytical methods to demonstrate that all degradation products are sufficiently detected and any unknown peaks are not interfering with the analyte peak. It correlates the estimated loss of a main analyte to the measured raise in the quantity of degradation products (Riley and Rosanske, 1996; Kirschbaum, 1988) Regulatory agencies use mass balance study to check the stability-indicating capability of analytical method and to check all the degradants are accounted for. In mass balance study the loss of parent drug is determined from sample assay and the increase in degradation impurities is measured from the related substance method. If the loss in assay value can be reasonably accounted for by the amount of degradants measured, then the mass balance is achieved. According to ICH mass balance is summation of the assay value and % of DPs to closeness to 100%. In principle, the influence of each stress degradation factor is first individually explored, then cross-influences are evaluated. The duration of the study will depend on the studied factor and the sensitivity of the product to that factor. Influence of different factors are discussed in section

40 ACCURACY Accuracy is the degree of sameness of test results acquired in a test method to the true results. In general actives or impurities are spiked typically at 50, 75, 100, 125, and 150% of their target concentration levels LIMIT OF QUANTIFICATION The limit of quantification can be defined as the smallest concentration of sample which gives a response which can be established with acceptable precision and accuracy. There are different types of approaches are acceptable according to ICH to identify the limit of quantification. Visual evaluation method: This method can be applied for instrumental and noninstrumental methods. LOQ is evaluated using known concentrations of sample and to get minimum lowest level with acceptable accuracy and precision. Signal-to-noise method: This approach can be applied to display base line noise. A typical signal-to-noise ratio is for quantification limit10:1 and 3: 1 for detection limit LIMIT OF DETECTION It is least level of sample which gives a measurable response but not need to be quantitated. LOD can be determined by Visual evaluation method and Signal-tonoise method. The analytical methods are validated which are able to quantify potential DPs and impurities with a LoD and LoQ at least as sensitive as the ICH threshold LINEARITY AND RANGE The linearity of the method proves the proportionality between concentration and response of the sample. For establishing linearity 5 minimum concentrations are recommended. 40

41 The range is the highest and lowest concentration for which the analytical method has acceptable precision, accuracy and linearity RUGGEDNESS The ruggedness of a method performed to get reproducibility when the method is executed under real conditions in different labs, different analysts, columns, of reagents, solvents source, and instruments. The results are compared with all the experiments with the %RSD values. As per ICH, the term ruggedness is not separately described; however ICH covered the topic in precision instead of ruggedness separately ROBUSTNESS The robustness is degree of ability of method to remain unchanged with small, measured deviations in the method factors BENEFITS OF METHOD VALIDATION The major advantage is it constructs the confidence for the developer and to user. As it looks costly and time consuming, but eradicates duplications ISOLATION TECHNIQUES It is often necessary to isolate impurities for their structural elucidation and qualification. Isolation entails removal or collection of the compound of interest from the other compounds present in a mixture. Further purification is achieved based on the compound s intended use. A successful purification protocol accounts for through put of crude as well as purity and yield of the targeted sample. A list of techniques employed for isolation is listed below. (i) Solid phase extraction (ii) Liquid-liquid extraction (iii) Accelerated Solvent extraction (iv) Supercritical fluid extraction (v) Column chromatography (vi) Preparative HPLC (vii) Preparative TLC and (viii) Flash chromatography (ix) High performance counter current chromatography (HPCCC). The selection of an optimum method for isolation of impurities is dependent on a number of considerations, the foremost being the method that was initially used to find the impurity. If the method is not too cumbersome to carry out and the 41

42 projected amount of the isolated impurity can be handled by the methodology in place, that method is obviously the preferred technique. If the amount of impurity needed for characterization, pharmacologic studies or toxicological studies is much greater, it may be necessary to rely on simple separation methods such as column chromatography, flash chromatography or TLC, because of their simplicity, flexibility, speed of analysis, and unique detection methods for both qualitative and quantitative analysis. Because of the relatively low levels of the impurities (and also in many cases, their close resemblance to the main compound), conventional separation techniques are usually not successful. The resolving power of chromatography is needed for this isolation. Since preparative HPLC was employed in the present investigation, this technique is described in detail as below PREPARATIVE HPLC Preparative HPLC is the most powerful and commonly used isolation and purification technique in the pharmaceutical industry (Verzele and Dewaele, 1986). Application of this technique comes into picture when the identification of impurity cannot be carried by use of simple analytical (chromatographic, spectroscopic and hyphenated) techniques. In this case, preparative HPLC isolation followed by spectroscopic investigation provides an appropriate solution for characterisation of impurity. In order to carry out successful isolation of targeted impurity, an appropriate analytical LC method needs to be developed for its detection. The HPLC and TLC information is very important for development preparative HPLC. The term Preparative chromatography is ambiguous and its meaning will often depend on its use. However, all preparative separations contain the collection of target and not interested with quantitative estimation and time retention measurement. Table 1.19: Comparison of analytical and preparative HPLC 42

43 APPLICATION AREAS OF PREPARATIVE HPLC Preparative HPLC is applied to isolate and purify valuable required products in the pharmaceutical, chemical as well as in biochemistry and biotechnology fields. The amount of sample to isolate or purify differs on the working areas. Its scale is depends on the amount of target to be purified. The working fields for preparative HPLC are described in Table Table 1.20: Working areas of preparative HPLC Compound amount Working area Milligram Isolation of enzymes Biological and biochemical testing Structure elucidation and characterization of - Side products from production - Metabolites from biological matrix - Natural products Gram Reference compounds (Analytical standards) Compounds for toxicological screenings - Main compound in high purity - Isolation of side products Kilogram Industrial scale, active compounds, drugs Figure1.13: Results of a preparative HPLC run High through put High purity Optimized 43

44 PREPARATIVE HPLC METHOD DEVELOPMENT Before staring preparative HPLC, below considerations must be taken which are described in Table Table 1.21: Optimisation parameters in Preparative method S.No. Required Parameters Considerations 1 Timelines Important in continues process Onetime requirement 2 Quantity <100mg for characterisation Grams level for reference standard 3 Purity <95%, more isolation options >99%, more challenging 4 Nature of feed RRT of target, level of target, solubility of feed Apart from that, below strategy (Figure1.15) is considered as a systematic approach. Figure1.14: Strategy for Preparative separation Selection of mode of chromatography: There are different chromatography techniques are available. Selection of suitable mode of chromatography is showed in Figure 1.15 and Figure

45 Normal phase mode Reverse phase mode Ion Exchange chromatography Chiral Specialty Figure 1.15: Modes of Chromatography Figure 1.16: Modes of HPLC 45

46 Achiral impurity isolations: For achiral impurities, the screening normally starts with the analytical separation method. If the stationary phase is available in either large columns or bulk and the impurity is well resolved from the other components in the mixture, this might be a good system for preparative use. The ideal preparative mobile phase would have no additives. If mobile-phase additives are required for separation, then use of a volatile additive (i.e., trifluoro acetic acid, acetic acid, triethyl amine, etc.) is preferred over a non-volatile additive (phosphate buffers) if removal of the mobile phase by evaporation or boiling is required. Finally, oxidizing buffers (per chloric acid, etc.) should be avoided if possible because of safety considerations with their use in large-scale processes. Often it is necessary to make adjustments in solvent strength and separation selectivity when developing or scaling up a preparative LC separation from analytical LC method. In many cases, only slight adjustments of one or two parameters like solvent percentage, ph change, and gradient change will be required to obtain the desired resolution for the peak of interest. It is important to note, however, that solely increasing or decreasing the strength of the mobile phase typically does not increase the resolution between the components; it only alters the retention of all the components. The resolution is frequently affected by adjusting the ph of the mobile phase, however; and occasionally simply substituting one buffer for another alters the resolution and retention. A thorough examination of the effect of ph on the retention/resolution can be achieved by examining three or four ph values (high, neutral, medium low and low). It is also important to know the ph range under which the column is stable. If the resolution between the impurity and other components in the mixture is too low then other stationary phases should be examined. Chiral impurity isolations: For situations in which the impurity is an enantiomer, the choice of stationary phase will be limited to chiral stationary phases. For a typical screen, the four common Daicel phases (Chiralcel OD, OJ, and Chiralpak AD, AS) are tried first and followed by the further trail on immobilized phase columns like IA,IB,IC available from Diacel. The initial screen for these columns is done with mixtures of ethanol or isopropanol in hexane or heptane. If resolution is found in this screen, the system can be optimized by examining other solvents; however, the choice of solvents is limited 46

47 by the chemical compatibility of these phases. If one of these phases does not resolve the desired compound, then screening moves to other phases OPTIMIZATION OF SCALE UP PARAMETERS Scaling up to Preparative HPLC - Sample load - Flow rate - Gradient Optimization SAMPLE LOAD The sample mass capacity of a column is directly proportional to the amount of packing material. Assuming that the same packing material will be used in the preparative column, scale the sample load according to the internal volumes of the columns, as follows (D prep) 2 X L prep Load prep = Load Anal.X ( D Anal) 2 X L Anal Where: D = Internal diameter of the column (cm) L = Length of the column (cm) Practically two types of sample load are used. 1) Volume overload 2) Mass overload (concentration overload) Volume overload: In volume overload peak position will shift to higher retention but start of the peak remains in the same position (Figure 1.17) unless injection in a weaker solvent. 47

48 Figure 1.17: volume overload Mass overload: In mass overload peak position will shift to lower retention but end of the peak remains in the same position (Figure 1.19). Figure 1.18: Mass overload Overloading depends on application, especially on the solubility of the crude which results in high throughputs. Concentration overload can be done effectively if the solubility of the crude is more than 20mg/mL. Otherwise volume overload or combination of both can be done. 48

49 FLOW RATE Assuming that: 1) Same Column packing material 2) Same Particle size material is employed To scale up the flow rate for preparative separations (D prep) 2 F prep = F Anal X ( D Anal) 2 Where: F = Flow rate (ml/min) D = Diameter of column (cm) GRADIENT OPTIMIZATION The system volume in the small-scale analytical system corresponds to a few column volumes. In the large-scale prep system, the system volume is a fraction of the column volume. This difference affects how soon the gradient changes reach the column. In the Analytical system, several column volumes must pass through the column before the gradient reaches the column. In the prep system, the gradient reaches the column almost immediately. If the gradient duration is same: Gradient duration large = Void volume large X Flow rate small Gradient duration small Void volume small X Flow rate large FRACTION COLLECTING METHODS The targeted peaks can be collected by different ways as described below. Manual fraction collection: In this method the desired fraction is collected manually from detector outlet. Automated fraction collection: In this type, fraction collections can be automated by the use of liquid handlers and software programme based on different parameters illustrated in Figure

50 Figure 1.19: Automated fraction collection parameters POST ISOLATION PROCESSING The collected fractions should be monitored for purity confirmation. Pool the desired purity fractions and target them to different post isolation procedures like solvent evaporation or liquid- liquid extraction or lyophilization to get the solid material COST REDUCTION METHODS It is always good to develop methods using less % of solvents as the preparative isolation approach frequently involves high cost. Develop shorter run time methods wherever feasible. Purify the crude (Max possible) before going for separations. Do staggered injections wherever possible. Inject as much load as possible without compromising the purity aspects. Optimize the method with respect to purity, yield and throughput. Adopt solvent recycling procedure wherever applicable TIPS FOR SUCCESSIVE PREPARATIVE ISOLATION PROCESS Inject more concentration (concentration over load) than more volumes (volume over load). Achieve maximum resolution between the target and its neighbour. Do extensive study on mass overloading and volume overloading. Under particular cases column dimensions can be varied to excess the load which leads to high productivity. Use non buffer conditions to avoid the purification steps, normal phase methods are preferable. 50