2. PHARMACEUTICAL ANALYSIS AND QUALITY CONTROL Ashraf M. Mahmoud, Ph.D. 1

3. Essential books and Text books  Vogels Textbook of Quantitative Inorganic Analysis, 6 th Edition Longman Scientific and Technical, USA (1998).  Christian G. D., "Analytical Chemistry ", John-Wiley and Sons, Inc New York (1994).  D. A. Skoog and d. M. west, "Fundamentals of Analytical Chemistry", 7 th ed CBS Publishing Asia Itd (2000). 2 Essential books and Text books

4. I. Development of analytical quality control laboratory and analytical sampling  Introduction to quality control  What is meant by quality ?  Quality control and quality assurance  Good manufacturing practices (GMP)  What is the goals of quality control?  Development of analytical control laboratory  Drug quality surveillance programmes  Analytical sampling according to WHO guidelines  Analysis of pharmaceutical products  Good chemical storage 3

5. Overview:  Quality control means different things to different people according to the field of interest.  Quality control is the responsibility of everyone in an enterprise.  Quality control in medicine is an old concept. As long as there has been a medication, there has been the need to control the quality of its performance. In ancient times, original reasons were to prevent Fraud and adultration of expensive materials with cheaper alternatives, which may have some medicine action, but mostly not.  Since the 20 th century other factors have come into consideration. Most important is the development of synthetic drugs via the fine chemical and pharmaceutical industries. Besides having the ability to cure or relive symptoms, modern drugs may be toxic by itself and/or contain toxic impurities by virtue of their manufacturing methods, reagents and intermediates. Introduction to Quality Control 4

6. All successful manufacturers tend to produce drugs that can be sold. Such manufacturers have a range of perfect products to suit their customer’s need and attract their confidence. Quality is the degree or grade of excellence possessed by an item Quality is the characteristics of a product or service that bear on its ability to satisfy stated or implied needs. Quality is a product or service free of deficiencies. Quality has no specific meaning unless related to specific function or object Quality must be built into the product during research, development, and production of pharmaceuticals. i.e. the quality is an intrinsic characters of the finished products Quality attributes in operations management What is meant by quality ? 5 Cost Quality DependabilitySpeedFlexibility

7. The suitability of drugs for their intended use may be determined by:  Their efficacy weighed against safety to healthers according to the label claim  Their conformity to specifications regarding identity, purity, potency, uniformity, bioavailability, and stability.  Identity: The correct active ingredient is present  Purity: The drug is not contaminated with potentially harmful substances  Potency: The correct amount of active ingredient is present (usually 100 ± 10% of the labeled amount)  Uniformity: consistency, color shape, and size of dosage form do not vary any when and any how  Bioavailability: speed and completeness of entrance of the used drug to the blood stream  Stability: The activity of the drug is guaranteed until the expiration date The characteristics and features of the drugs which determine its quality 6

8.  There is often confusion over the meaning of quality control and quality assurance.  The proper definition of the terms can be found in International Organisation for Standardization (ISO), European Standard (EN) or British Standard (BS) Quality assurance means; (prevention of defects) All those planned and systematic actions necessary to provide adequate confidence that a product or service will satisfy given requirements for quality All planned activities designed to ensure that the quality control activities are being properly implemented. Quality control means; (Detection of defects) All the operational techniques and activities that are used to fulfill the requirements for quality All the planned activities designed to provide a quality product. Quality Control and Quality Assurance 7

9.  GMPs are the minimum requirements that the industry must meet when manufacturing, processing, packing, or holding drugs for human and veterinary use. Good Manufacturing Practices (GMP) 8 Quality Control is part of Quality Assurance GMP is part of Concerned with sampling, specifications, testing, organization, documentation & release Takes place within a Quality Management system Concerned with production & quality control

10. To assure that the drug products in the markets comply with the registration requirements in terms of safety, efficacy, and quality. To ensure compliance to pre–determined standards - approved indications, packaging and labeling requirements. To ensure the stability of the active ingredients in the finished products. To assure that the drug products contain the labeled amount of the active constituent(s) within the accepted limits Objectives of quality control What is the goals of quality control? 9 The extent of the routine drug quality control, carried out by the National Drug Regulatory Authorities will depend on: Capacity of the national drug QC lab. Extent to which the quality of the product has been assessed prior to registration Extent to which the requirements for GMP are implemented Number of products imported from abroad

11. WHO has produced WHO certification scheme on the QC of pharmaceutical products moving in International Commerce which is very useful for the importing countries. WHO Certification Scheme for Products moving in International Commerce is an international voluntary agreement, created to enable countries with limited drug regulatory capacity to obtain partial assurance from exporting countries concerning the safety, quality and efficacy of the products they plan to import. http://www.who.int/medicines/areas/quality_safety/regulation_legislation/certification/en/ On this website you can find information about:  competent authorities participating in this certification scheme.  model certificate of a pharmaceutical product.  guidelines on the implementation of this certification scheme. QC does not consist of few analytical procedures carried out by a laboratory at the end of a production run, but is a series of procedures which start at the inspection of plans for a new product and continue well beyond the dispatch of each batch. WHO Certification Scheme 10

12. Quality Management: It is the aspects of the overall management that determines and implements the quality policy. Quality Policy: It is the overall quality intentions and directions of an organization with respect to quality as formally expressed by the top management. Quality Systems: The ISO, EN and BS standards define a quality system as; the organisational structure, responsibilities, procedures, processes and resources for implementing quality management. Briefly the quality system is a combination of quality management, quality control and quality assurance. Quality Management 16

13. Aim It was constructed to achieve continuous good performance in all processes performing expected excellent productivity with significant perfection. GQM was firstly originated in Japan in 1940s during the postwar-reconstruction to control the quality of the products. It was also called Total Quality Control (TQC) Pricipal goals were: The continous improvement in quality through research and development (R&D), exceeding the customer needs, good personel management, and job security with understanding (know-how key) Good quality is to be built from the all beginning Realization of the Standard Operational Procedures (SOPs) Predominating the collaborative team work in all steps of the procedures understanding that “errors” are not always “mistakes” Open & transparent atmoshere → cooperative attitude i.e. workers look together for the source of variability and test together how to minimize or cure it Good Quality Management (GQM) 17

14. Figure shows the way may lead to a variability (example of constructing the Tunnel between France and England). 18

15. Good management’s job  Direct the efforts of all components toward the aim of the system.  Everyone in the organization must understand the aim of the system.  Everyone must understand the damage and loss to the whole organization from a team that seeks to become selfish and independent. “ Deming Cycle ” or “Shewhart Cycle” or “Deming Wheel” It describes a simple method to test information before making a major decision. It is an iterative 4-step problem-solving process called PDCA “Plan – Do – Check (study) – Act” Plan : design the experiment Do : performing the steps of experiment Check: check the results by testing information Act : act on the decisions based on those results Good Quality Management Cycle (GQMC) 19

16. Act Plan CheckDo PLAN: Establish the objectives and processes necessary to deliver results in accordance with the specifications. DO: Implement the processes (like the chemical analysis). CHECK (STUDY): Monitor and evaluate the processes and results against objectives and specifications and report the outcome. ACT: Apply actions to the outcome for necessary improvement. This reviewing all steps (Plan, Do, Check, Act) and modifying the process to improve it before its next implementation. Deming Management Cycle 20

17.  Good analytical practice (GAP) and good laboratory practice (GLP) are concepts concerning with the building of good quality in analytical practice.  Many compendia and authorities, such as the United States (USP), British (BP), Europian (EP) Pharmacopeias and the Association of Official Analytical Chemist's (AOAC), collect a variety of full validated analytical methodologies and techniques.  Academic analysts seem to avoid discussions about validations; as they develop and optimize analytical methods using mostly pure standards. Therefore, many analytical methods lacking full or adequate validation have been appeared in open literature. These methods, in practice, may never be useful for samples encountered in complex matrices.  In order to perform a GAP or GLP, full validated analytical methodologies and techniques must be applied. GOOD ANALYTICAL PRACTICE (GAP) & GOOD LABORATORY PRACTICE (GLP) 21

18.  Calibration is conversion of an observed value into a more reliable result, which is called "corrected", "true" or "calibrated” value  How ? By using a reference standards in a similar matrices Calibration in chemical analysis 22

19. Analytical technique: is the scientific principle adopted to one or more instruments to obtain some required informations about materials; such as gravimetry, volumetry or instrumental methods of analysis. Analytical process: is a series of operations between sampling and results, including preliminary steps and measurement followed finally by data treatment and handling. Analytical method: is actual application of a specific analytical technique in the analytical process. Analytical protocol (procedure): is a set of precise instructions (similar to orders) followed in an analytical method in order to determine one or more analytes in a given sample. The term protocol seems to be more specific than procedure. Definitions 23

20. 1.The Organization for Economic Co-operation and Development (OECD),which has developed the Good Laboratory Practice "GLP” 2.International Conference on Harmonisation (ICH guidelines) of technical requirements for registration of pharmaceuticals for human use; validation of analytical procedures. 3.The International Organization for Standardization (ISO) has produced a rang of standards and guidance relevant to laboratories. 4.The European Standards (EN-series). 5.The European Committee for Standardization (CEN) has produced its own range of standards concerning quality. 6.The British Standards Institution (BSI) has also produced a range of standards addressing quality. Standard Quality Systems for Analytical Laboratories 24

21. It consists of at least 2 primary units 1.Analytical control laboratory or unit (ACL or ACU) 2.Inspection control unit (ICU) Function of the analytical control unit It is responsible for testing and approving or rejecting raw materials (active or not), packaging, work in-process control and finished product. It must be: 1. Staffed with trained personnel experienced to perform complex analysis required to evaluate the acceptance of a product. 2. Well equipped with instruments which allow accurate analysis. 3. Detailed specifications of test methods must be available. The specification must include the limits for acceptance or rejection for each parameter. Quality Control Unit 25

22. Laboratory setup  Constitution of pharmacy administration to provide the required administrative framework.  Define the objectives (the drugs to be tested and the types of tests to be applied)  Sampling programme Units of ACL 1.Physico-chemical investigation unit 2.Physical criteria specification unit 3.Stability indication unit 26Development of Analytical Control Laboratory (ACL)

23. 1.Accommodations (Rooms for chief analyst, consultants, general office registration, documentation and sample rooms, and stores (chemicals, glassware, spares and consumable of instruments) 2.Laboratory furniture and fixture 3.Chemicals and glassware 4.Books and journals 5.Staffing and manpower development 6.Ancillary staff (efficient system of documentation is essential) 7.Problems and constraints 8.Laboratory organization and administration DEVELOPMENT OF PHASES (STAGES) 30

24. Sampling according to WHO guidelines for sampling of pharmaceutical products and related materials 1.purpose of sampling? 2.type of tests intended to be applied to the samples? 3.type of products/materials to be sampled? 4.are the sampling facilities adequate? 5.are the responsibilities of the samplers clear?  Sampling process A.Preparation for sampling B.Sampling operation C.Sample storage and retention 31  Sampling programme  What is the sampling ?  Types of samples What............?  Before sampling:

25.  Sampling; is the process of selecting a portion of material to provide information about a larger body of material.  National Measurement Accreditation Service (NAMAS) defines sampling as “ A defined procedure whereby a part of a substance ( matrix, material or product ) is taken for testing to provide a representative sample of the whole  Sample: is a representative portion selected from the bulk.  Analytical sample: is a small portion selected from the sample. Sampling 32

26.  The samples can be classified according to the type of material into: A) Bulk materials; e.g. Statring materials & Natural products B) Intermediates in the manufacturing process. C) Finished products D)Containers, Packaging materials and labelles.  The samples can be classified according to the physical state into: 1. Gas 2. Liquid 3. Solid.  The samples can be classified according to homogeneity into: 1.Homogenous materials as Liquid preparations 2.Heterogeneous materials as solid dosage forms (powders, granules and vegetables)  The samples can be classified according to the sampling plan into 4 types: 1.Representative sample Is typical of the parent material for the characteristic under inspection. Is easy to collect from homogeneous bulk. For heterogeneous bulk, care should be taken to overcome problems of mixing variation and separation Sample may be representative if the concentration of analyte is at 5% w/w Types of samples 33

27. 2. Selective sample Is deliberately chosen by using sampling plan that screens out materials with certain characteristics or selects only material with other relevant characteristics. This may be called directed or focused sampling. 3. Composite sample It consists of two or more portions of material (collected at the same time) selected to represent the material being investigated. The components of the sample are taken in proportion to the amount of the material they represent. Advantage: reducing the cost of analysing large numbers of samples. 4. Random sample It is selected by a random process to eliminate questions of bias in selection and to provide a basis for statistical interpretation of measurement data. The sample is selected so that any portion of the material has an equal (or unknown) chance of being chosen. There are three types of random sampling: Types of samples (cont.) 34

28. a. Simple random sampling;  In which any sample has an equal chance of selection.  Example: Samples selected from the population are given numbers in such a way that all the numbers have the same number of digits. Random numbers are then read off from the random table starting at a random point and the corresponding member of the sample population.  Advantage: all the members of the population have an equal chance of being selected.  Disadvantage: labor time consuming. b. Stratified random sampling In which the lot is subdivided/stratified and a simple random sample selected from each stratum. Types of samples (Cont.) 002207996490765 030593666444805 35

29. c.Systematic sampling In which the first sample is selected at random, then the subsequent samples are taken according to a previously arranged interval e.g. every 5 th, 10 th or whatever is appropriate. Advantages: The simplicity and time saving. Disadvantage: It is biasing to some extent. Continuous systematic sampling; An automatic system may be used to provide the portions of material for analysis regularly. Types of samples (Cont.) 36

30.  Sampling tools should be available to the sampler, e.g. to open containers (knives, hammers,...), material to reclose the packages (sealing tape), self- adhesive labels to indicate that some of the contents have been removed, etc...  Sampling tools should be made of inert materials (e.g. polypropylene or stainless steel; avoid glass) and kept very clean. After use, thoroughly washed, rinsed with water or suitable solvent, dried and stored in clean conditions.  Disposable sampling materials can also be used.  Washing facilities should be located in, or close to, the sampling area.  Cleaning procedure should be documented and validated (= demonstrated efficiency).  Sterile pharmaceutical products should be sampled under aseptic conditions. 38 Sampling tools

31. Make sure that representative samples are taken in sufficient quantity. Samples should never be returned to the bulk. Sample collection form: written record (documentation) of the sampling operations, always kept together with the collected sample, it includes;  batch number,  sampling date/place,  reference to sampling protocol used,  description of containers and materials sampled,  possible abnormalities,  name/signature of the sampler. Take into account previous experience with the product and supplier. 41 Sampling process

32. Sampling for acceptance: the degree of acceptable risk is calculated by using the sampling plans to explain the probabilities and risks. Acceptance of sampling: can be determined either by attributes or by variables. 1.In sampling by attributes The item in the batch of product either conforms or not. The number of nonconformities in the batch are counted and if this reaches a predetermined figure the batch is rejected. 2. In sampling by variables The characteristic of interest is measured on a continuous scale. If the average meets a predetermined value and is within an acceptable standard deviation, the batch is accepted. 42 Sampling for acceptance

33.  Acceptance Sampling Plan:  These sampling plans consist of a sample size and a decision rule.  The sample size is the number of items to sample or the number of measurements to take.  The decision rule involves the acceptance limit(s) and a description of how to use the sample result to accept or reject the lot.  Design Method: To design a sampling plan by the two-point method, the designer specifies two points on the Operating Characteristic Curve (OC- Curve). Sampling plans 44  These two points define the acceptable and unacceptable quality levels for the purpose of acceptance sampling.  The two points also determine the risks associated with the acceptance/rejection decision.

34.  Advantages of Sampling Plans: 1.Discrimination: The sampling plans will require enough testing and inspection to discriminate between acceptable and unacceptable quality levels. 2.Reduce Cost: They will save you time and effort by using no more testing and inspection than necessary in order to minimize cost. 3.Explanation of the probabilities and risks: They enable you to quickly know, describe, and explain the probabilities and risks using OC-Curve.  Examples for sampling plans for starting materials: 1.The “n-plan” or normal plan It is Only used when material is considered uniform and from a recognised source. 2.The “p-plan” or pooling plan May be used when material is considered uniform, from recognised source and the main purpose is to test for identity 3.The “r-plan” or random plan May be used when material is considered non-uniform and/or obtained from a not well know source. Can be used for herbal medicinal products used as starting materials. 45 Sampling plans (cont.)

35.  Tablets sample reduction is normally carried out by taking at least 20 tablets, they are counted and weighed then the average weight of one tablet is calculated. Tablets are grinnded into fine powder and an accurate weight equivalent to one tablet is taken for analysis. Obtained resuls will be results of the average tablet, which is not providing information about the variability among tablets. Subsampling 49

36.  Quality of sample; Properties of the analyte such as volatility, sensitivity to light, thermal stability and chemical reactivity all have to be considered in designing a sampling strategy. These factors need to be taken into account to ensure that the quality of the sample does not degraded before the measurements are made.  Sample size; How large a sample do you need? This is the question you need to ask yourself before the samples are collected; The minimum sample size can be determined using the concept of sampling constant. Sampling constant K s It is the sample weight necessary to ensure a relative sampling error of 1% in a single determination. Value of √K s is numerically equal to coefficient of variation for results obtained on a 1g sub-samples in a procedure free from analytical error. 50 Sampling process

37.  Store the sample in a properly labelled container: sample type, name of material, identification code, batch number code, quantity, date of sampling, storage conditions, handling precautions, container number...  Containers  Containers used to store a sample should comply with the storage directions for the active pharmaceutical ingredient, excipient or drug product: 1.should not interact with the sampled material. 2.should not allow contamination. 3.should protect the sample from light, air and moisture. 4.should be sealed and adequately labelled. 5.avoid mix-up when containers are opened (screw caps, separate lids). 6.manipulations/unauthorised opening should be easy detectable. 7.transported in such way as to avoid breakage. Sample storage and retention 51

38. Introduction to Pharmaceutical Analysis After sampling operation is completed successfully, now analysis operations will be started. The chemical analysis provides the methods needed for answering three basic questions about a material sample? In what form is present? Where is present? How much? Before starting work on a sample, it is vital to enquire why the work is being done, what will happen to the results, what decisions will be taken depending on the results obtained. 54

39. Areas of Pharmaceutical Analysis and Questions They Answer  Detection: Does the sample contain substance X?  Quantitation: How much of substance X is in the sample?  Identification: What is the identity of the substance X in the sample?  Separation: How can the species of interest (substance X) be separated from the sample matrix for better quantitation and identification? 55

40. An analysis involves several steps and operations which depend on: 1.the particular problem 2. your expertise 3. the tools or equipments available. An analysis involves several steps and operations which depend on: 1.the particular problem 2. your expertise 3. the tools or equipments available. 56 Selection and development of the analytical method

41. Steps of chemical analysis 1. Define the problem (What is the purpose of an analysis?) Factors What is the problem – what needs to be found? Qualitative and/or Quantitative What will the information be used for? Who will use it? 1.To prepare a databank of figures to establish trends. 2.To accept or reject a chemical or product before use in a manufacturing operation. 3.Assessment of the value of a consignment of product before payment. 4.Prosecution of a company for selling a product not up to the stated specifications. 5.Criminal charges of an individual found to be in possession of drugs. What is the budget? The problem solver (The analyst) 57

42. ©Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) Qualitative analysis is what. Quantitative analysis is how much 58

43. 2. Select the method (Factors to be considered in choosing an analytical method) After defining the problem and the purpose of an analysis, you must now decide which particular analytical procedure to choice. The choice of a method could be justified depending on certain factors. What factors will influence your choice? 1.Type and size of the sample & throughput. 2.Conc. range of the analyte to be determined. 3.Sensitivity of the method and limits of detection & determination 4.The required degree of accuracy and precision. 5.Speed of analysis and its convenience with the purpose. 6.Tools and instruments available. 7.Cost. 8.Safety measures 9.Selectivity. 10.Availability of standard methods 11.Analyst experience Steps of chemical analysis (cont.) 59

44. © Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) Different methods provide a range of precision, sensitivity, selectivity, cost and speed capabilities. 60

45. ©Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) The sample size dictates what measurement technique can be used 61

46. 62 3. Obtain a representative sample Factors to be considered 1.Type and size of the sample. 2.Homogenity of the sample 3.Sampling errors. 4. Sample preparation for analysis Factors to be considered 1.Type of the sample (solid, liquid or gas) 2.Dissolve, ash or digest the sample 3.Is a chemical separation or masking of the interferences needed? 4.The need to concentrate the analyte 5.The need to change or derivatize the analyte for detection 6.The need to optimize the conditions (pH, reagent conc., etc) Steps of chemical analysis (cont.)

47. 63 Steps of chemical analysis (cont.) 5. Perform any necessary chemical separation 1.Distillation 2.Precipitation 3.Solvent extraction or solid phase extraction 4.Chromatography or Electrophoresis (as a part of measurement step) 6. Perform the measurement 1.Calibration 2.Validation, control, blank 3.Replicates 7. Calculate the results and report 1.Statistical analysis (reliability) 2.Report the result with limitation and accuracy informations

48. Before considering how one can ensure that analytical data obtained are correct and fit for the purpose required, it is worth thinking about what could go wrong. Then it will be easier to work out how to avoid making mistakes. Reasons for incorrect results 1- Incompetence e.g. mistakes in calculations, poor laboratory practice, errors in labeling of samples and equipment used in subsequent analysis. 2- The used method e.g. 1.Use the method outside the tested calibration range. 2.With matrices that were not included in the original validation process. 3.Introduction of subtle changes in the procedure to suit the analyst circumstances or convenience. 4.Changes to the recommended purity of reagents used can also influence the results obtained. Reasons for incorrect analytical results 64

49. 3- Contamination due to laboratory environment a.Common reagents or solvents present in the laboratory. b.It is also important to ensure that your colleagues working nearby are not using chemicals which could affect your determination. 4- Interferences The method chosen must discriminate between the analyte of interest and other compounds also present in the sample. 5- Calibration Normal QC/QA procedures require all the instruments, glassware, ovens, water baths, balances..etc to be calibrated against traceable standards at regular intervals. 6- Sampling No analysis, however carefully carried out will be accurate unless the test sample taken for analysis is truly representative of the bulk material. 65 Reasons for incorrect analytical results (cont.)

50. 7- Loss and degradation Analyte may be lost at various stages of the analytical procedure for a number of reasons: 1.Degradation by heat, oxidation..etc. 2.Loss due to volatility during digestion or evaporation. 3.Loss resulting from adsorption on surfaces. 4.Incomplete extraction of the analyte from the matrix. (Note that) This part will be discussed again under the systematic errors due to analytical methods. 66 Reasons for incorrect analytical results (cont.)

51. Errors Error is defined as the difference between an individual result and the true value of quantity being measured. Types of errors a) Random errors (Indeterminate) b) Systematic error (Determinate) (A) Random errors (indeterminate error): 1.Occur due to uncertainties in a measurement. 2.It is difficult to discover their origin (the sources of these errors cannot be positively identified). 3.The magnitudes of individual random errors are not measurable. 4.The most important consequence of random errors is they cause data from replicate measurements to fluctuate in a random manner on both sides of the average value. 5.The scattering of individual measurement around the mean value is a direct indication of indeterminate-type error. 67

52. (B) Systematic error (Determinate error) 1.Systematic errors have a definite value and an assignable cause. 2.In principle (But not always in practice) the analyst can measure and account for these errors. 3.A determinate error is often unidirectional (All of a series of replicate analyses to be either high or low). 4.The magnitude of the systematic error can be determined. 5.It is not possible to list all causes of determinate errors. limitations to both precision and accuracy can be traced to three general sources: 1)Instrumental errors: is attributable to imperfection in measuring devices. Measuring devices (e.g. pipettes, burettes and volumetric flasks) are potential sources of determinate errors due to the use of glassware at temperatures that differ significantly from the calibration temperature, distortion in the container walls due to heating, errors in the original calibration or contaminations on the inner surfaces of containers. Types of errors 68

53. Instrumental errors (cont.) Errors of this type are readily eliminated by re-calibration. Also measuring devices powered by electricity are commonly subjected to determinate errors, due to decrease in voltage, increased resistance in circuits because of dirty electrical contacts, temperature effects on resistors….etc. Again these errors are unidirectional, detectable and correctable. 2) Analytical methods error Discussed before under reasons for incorrect analytical results. Errors are inherent in the method are frequently difficult to detect and are thus the most serious of all types of determinate errors. 3) Personal errors This type of errors is introduced by the analyst due to carelessness or lake of experience. Color blindness or other physical handicaps often increase the probability of personal errors. Types of errors (cont.) 69

54. Examples of these errors are false judgment of colors at the e.p. of a volumetric analysis, false estimating the position of a pointer between two scale divisions of an electrochemical device and false reading of a liquid level with respect to the graduation in a pipette or burette. A near-universal source of personal errors is prejudice or bias. Most of us have a natural tendency to estimate scale readings in a direction that improves precision in a set of results. Gross mistakes: It is a type of personal errors. Errors of this type include arithmetic mistakes, transposition of numbers in recording data, reading a scale backward, reversing a sign or using a wrong scale. These errors are ordinarily the consequence of carelessness and can be eliminated by care and self-discipline. Types of errors (cont.) 70

55.  Determinate errors may be either constant or proportional errors. Constant error 1.is independent of the size of the sample e.g. the small amount of standard remained after the color change in a volumetric analysis. 2.It approximately remains constant regardless of the total volume of standard consumed for the titration. 3.Therefore, the relative error of this type will be more serious as the total volume of sample decreases. Proportional errors 1.increase or decrease in proportion to the size of the sample taken for analysis, e.g. interfering contaminants in the sample. 2.The relative error of this type will be independent of the sample size. Types of errors (cont.) 71

56. Errors can be minimized by using one or more of the following: 1- Calibration of equipment & application of corrections. All apparatus and glassware should be calibrated periodically and the appropriate corrections applied. 2- Running a blank determination. Carrying out the experiment under the same procedure condition without adding sample. Blank is carried out to determine the effect of all the constituents of experiment other than the sample on results. 3- Running a control determination. Carrying out the experiment under the same procedure condition using a quantity of a standard substance which contains the same weight of the constituent as is contained in the sample. Weight of the constituent in the sample can then calculated from the relation; Minimization and correction of errors 72

57. 4- Using of independent methods of analysis In some instances the accuracy of a result may be established by carrying out analysis by an entirely different independent method. 5- Using standard addition method  A known amount of the constituent being determined is added to the sample, which then analyzed for the total amount of constituent present.  The difference between the analytical results for samples with and without the added constituent gives the recovery of the amount of added constituent.  Satisfactory recoveries give confidence in the accuracy of determinations. Minimization and correction of errors (cont.) 73

58. Concentration Absorbance Standard addition method 74

59. 6- Using of internal standards 1.It is of particular value in HPLC and GC analysis. 2.It involves adding a fixed small amount of suitable reference material (the internal standard) to a series of known concentrations of the material to be measured. 3.Ratios of the physical value (absorption or peak size) of internal standard and the series of known concentrations are plotted against the concentration values to give a straight line. 4.Then any unknown concentration can be determined by adding the same quantity of internal standard and finding ratio from graph. Minimization and correction of errors (cont.) 75

60. Concentration Peak hight ratio Calibration curve Internal standard method 0 10 5  g/mL 0 10 10  g/mL 0 10 25  g/mL 0 10 50  g/mL 0 10 75  g/mL 0 10 100  g/mL Internal Standard Compound 0 10 Unknown Analyte peak Internal standard peak 76

61. 7- Running parallel determinations These serve as a check on the result of a single determination and indicate only the precision of the analysis. Good agreement between duplicate and triplicate determinations does not necessarily justify the conclusion that the result is correct. 8- Amplification methods Used in determinations in which a very small amount of material is to be measured (Its response signals may be beyond the limits of apparatus available) e.g. HPLC equipped by spectrometric detectors. Minimization and correction of errors (cont.) 77

62. Results that collected from an analytical method called raw data and they need to be interpreted to give a significant judgment. For any assay to give a significant data need to be validated. A primary objective of the validation of analytical method is to establish the traceability to the recognized reference (pure substance). Traceability means that all the steps of analytical procedure should be performed and recorded keeping all essential information without introduction of any wrong information. Data Processing 78

63. II. METHOD DEVELOPMENT & VALIDATION  What is the validation of the method?  Relationship between validation and method development  Types of analytical procedures to be validated  Typical validation characteristics  Calibration model selection  Range, linearity, precision and accuracy  Limit of detection and limit of quantification  Selectivity and specificity  Analyte stability  Chemical purity and quality control  Sources of Impurities  Physical stability of pharmaceutical formulations  Chemical stability of drug products  Stability indicating assays 79

64. METHOD DEVELOPMENT (Optimization of Experimental Factors; pH, reagent, Temperature, Time of reaction ) Method validation experiments Calibration model Precision & Analyte Range & Linearity Accuracy Stability Collate results *METHOD VALIDATION Write Validation report * METHOD Apply Validation APPLICATION Relationship between validation and method development

65. Validation of the method: Validation of analytical method is the process of determining the suitability of methodology for the intended. Relationship between validation and method development: Validation is not a method development tool. Validation does not make a method good or efficient. Method development scientist should not enter the validation process. Validation process is a confirmation that the method is suited for its intended purpose

66. 66 TYPES OF ANALYTICAL PROCEDURES REGULATORY ALTERNATIVE STABILITY-INDICATING ASSAY USP-NF / BP / EuPharm Proposed, full validated method Equal or better than Regulatory method Rational of use; release, stability testing Provide evidence that the method is able to discriminate the major analyt from other Potential Impurities, Degradation Products, andexcipients, Types of Analytical Procedures

67. AIM OF THE ANALYTICAL METHOD (CATEGORIES) CATEGORY I Quantitation of major compound in bulk or finished form. CATEGORY II Det. Of IMPURITIES / degradation product(s) in bulk drug or in finished product. CATEGORY III Determination of performance characteristics ex: dissolution, drug release. CATEGORY IV Identification tests Types of Analytical Procedures to be Validated

68. Most common types of analytical procedures are :  Identification tests;  Quantitative tests for impurities' content;  Limit tests for the control of impurities;  Quantitative tests of the active moiety Identification tests Are intended to ensure the identity of an analyte in a sample via comparison of a property of the sample (e.g., spectrum, chromatographic behavior, chemical reactivity, etc) to that of a reference standard. Types of Analytical Procedures to be Validated

69. Testing for impurities Can be either a quantitative test or a limit test for the impurity in a sample. Different validation characteristics are required for a quantitative test than for a limit test Assay procedures Are intended to determine the amount of analyte present in a given sample. The assay represents a quantitative measurement of the major component(s) in the drug substance.

70. Typical validation characteristics which should be considered are : 1.Linearity & range 2.Accuracy 3.Precision (Repeatability, Intermediate Precision & reproducibility) 4.Detection limit 5.Quantitation limit 6.Specificity and selectivity It should be noted that robustness, ruggedness & system suitability are not listed in the items but should be considered at an appropriate stage in the development of analytical procedure. Typical validation characteristics FULL VALIDATION IS NECESSARY IF; 1.The method of synthesis changed from that of patented innovator’s method 2.Changes in the composition of the drug product (excipients, …etc), 3.Changes in the analytical procedure (critical change) 4.If the method is not cited in the compendia (USP/BP/EuPharm)

71. ANALYTICAL VALIDATION PARAMETERS [PERFORMANCE CRITERIA] SELECTIVITY / SPECIFICITY LINEARITY AND RANGE DETECTION LIMIT LOD QUANTITATION LIMIT (LOQ) ACCURACY ROBUSTNESSRUGGEDNESS PRECISION SYSTEM SUITABILITY ANALYTICAL VALIDATION PARAMETERS

72. 72 LINEARITY AND RANGE DEFINITION: Test results are directly or by a well- defined mathematical transformation, proportional to the concentration of analyte in sample within a given range. RANGE: Interval between the upper and lower levels of analyte, that have Been proved to be determined with suitable level of precision, accuracy, and linearity. Concentration, ng/mL Response, mVolt Least square regression equation: Y = A + B X

73. 73 Y = A + B X Y = Response in mVolt, or absorbance unit A = Intercept value from Y B = Slope (response, sensitivity) X = concentration (ng or ug, …) R = Regression co-efficient, should close to unity (0.9999-0.9980) Concentration, ng/mL Response, mVolt Concentration, ng/mL Response, mVolt High A value Low A value Low A value? Low B value ?

74. 74

75. 75 ACCURACY DEFINITION: The closeness of test results obtained by that method to the true value Expressed as % RECOVERY or Difference from mean DETERMINATION: 1.Accuracy should be established across the specified range of the analytical procedure (item # 2 below) 2.3 levels (conc), assay each 3 times = 9 determinations 3.Expressed as % recovery or difference from mean.

76. ASSAYIMPURITIES Drug Product Drug substance How to calculate the accuracy in the following cases: Calculate and compare the % Recovery, or %Difference SPIKE known amount RS: Apply the method using reference standard (RS) (known real amount) Compendial method: compare the amount found using the proposed and USP/BP method Calibration CURVE: compare the ESTIMATED values (CURVE) with the found values RS + Synthetic Mixture SPIKE USP method / compare CALIBRATION Curve

77. 77 DEFINITION: The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of a homogeneous sample PRECISION Expressed as: % RSD of assay (content found) of 6 repetitive determinations or 3 determinations of 3 levels (concentrations)

78. 78 ACCURACY AND PRECISION No Accuracy No Precision No Accuracy Precise Accurate Precise Precision may be considered at three levels: 1.Repeatability 2.Intermediate precision 3.Reproducibility

79. 1. Repeatability Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision. 2. Intermediate precision Intermediate precision expresses within- laboratories variations: different days, different analysts, different equipment, etc. 3. Reproducibility Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology).

80. 80 END POINT, mLmg, FOUND % RECOVERY 20.00 30.00 100.00 20.10 30.05 100.17 20.05 30.02 100.07 19.95 29.97 99.93 19.91 29.87 99.56 20.15 30.24 100.80 Assay of Phenelzine 30 mg tablets BP 2002 Mean 30.025 100.09 SD 0.1221 0.4063 %RSD 0.406 0.406 BP LIMIT: The RSD% value is NMT (Not More Than) 1.5%. Since the RSD did not exceed 1.5%, the method is precise

81. 81 Concentration, ng/mL Response, mVolt Precise, accurate Concentration, ng/mL Response, mVolt Lower precision, lower accuracy ACCURACY AND PRECISION

82. DEFINITION: The lowest amount of analyte in a sample that can be detected, but not necessarily quantitated VISUAL SIGNAL TO NOISE S/N = 3:1 DL = 3.3  / S From Calib. Curve DETECTION LIMIT (LOD)

83. 83 QUANTITATION LIMIT (LOQ / QL) DEFINITION : It is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy IMPORTANCE: Assays for low levels of compounds in sample matrices, such as impurities in bulk drug substances and degradation products in finished pharmaceuticals Determination SIGNAL TO NOISE S/N = 10 to 1 SD of Response QL = 10  / S From Calib. Curve VISUAL Acceptable precision (RSD%) & accuracy (%Recovery)

84. SPECIFICITY / SELECTIVITY DEFINITION OF SPECIFICITY : The ability of the analytical method to determine the analyte completely clear and definite in presence of other components that may be expected to be present (impurities, degradation products, matrix) A: Selective method / B: Non-Selective method A B DETERMINATION: If HPLC or GC: impurities and deg. products completely separated from major peak. Identify peak by Rt, resolution parameters. If TLC: R f factor of the major compound and other impurities.

85. DEFINITION OF SELECTIVITY : The term “selectivity” may also be used when the method is not specific for just one analyte but it is selective for a group of analytes having common properties. Lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedures. For example: Non-aqueous titration, although it is most commonly used in official assay methods, it is non specific. It is relatively good in other validation parameters. SPECIFICITY / SELECTIVITY

86. 86 RUGGEDNESS Analyst to Analyst DEFINITION: Degree of reproducibility of test results obtained by the analysis of the same samples under a variety of conditions. DETERMINATION: The method was RUGGED, because we got a comparable RSD% values when the method was repeated but using 1.Different analyst 2.Different Laboratory 3.Different Instrument 4.Different lot of reagent 5.Different assay elapsed time.

87. ROBUSTNESS DEFINITION: Is a measure of method capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. EXAMPLE:Method AMethod B Method parameters:RobustNot Robust pH = 10 (Ammonia buffer)± 1 or 0.4 Temp. = 40 degree ± 2 or 0.2 Centrifuge 10 minutes at 5000 rpm ± 3 or 1 0.2 mL of 0.1N HCl ± 0.1 or 0.02 Flow rate1 mL/min ± 0.2 or 0.1 DETERMINATION: Make slight change of the analysis conditions, and observe the analysis parameters (end point, retention time, resolution, intensity, recovered content)

88. Method Robustness In an HPLC experiment, the following representative parameters may be evaluated: HPLC manufacturer Lot-to- lot column variation Column supplier Flow rate Column temperature Mobile phase pH Ionic strength Detector wavelength Gradient slope Injection size, sample concentration ROBUSTNESS

89. 89 FOR USP/BP METHOD VALIDATION SYSTEM SUITABILITY NECESSARY If Different 1.Analyst 2.Instrument 3.Unknown Solution STABILITY System suitability testing ensures that total system is functioning at any given time. Coupled with previous instrument qualification, and method validation, provides assurance that the method is optimal for its intended use. DETERMINATION Repeat the analysis procedure at least 6 repetitive times, Calculate the %RSD for each result parameter (ex: Retention time, Resolution, Peak area)

90. ANALYTICAL REPORT Summary (Title, and aim of the work) Equipment (Manufacturer, calibration date) Material and analytical standards (Source) Chromatographic condition (Column, flow rate, pH, wavelength temperature, mobile system) Sample preparation Selectivity / stability indicating studies Forced degradation (for stability indicating assay) Linearity and range Limits of detection / quantitation Repeatability (system suitability), Accuracy and precision Ruggedness Robustness

91. VALIDATION DOCUMENTATION Consists of a protocol, test data, and a final report. Protocol may have data tables to enter test results, requiring only a short executive summary to summarize results and a reference or attachment of raw data. A copy of method procedure and a method development report are appended to the validation protocol. In general, validation protocol should contain the following: 1- Method principle/objective 2- List of responsibilities (laboratories involved and their role in the validation) 3- Method categorization according to ICH or USP or other methods. 4- List of reagents (including test lots) and standards required

92. 5- Test procedures to evaluate each validation parameter and proposed acceptance criteria 6- Plan or procedure when acceptance criteria are not met the requirements for the final report 7- Appendixes 8- Method development report 9- Method procedure The validation process cannot proceed until the protocol and all parties involved approve the acceptance criteria. VALIDATION DOCUMENTATION

93. REFERENCE STANDARDS REFERENCE STANDARD [1] COMPENDIAL RS [2] WORKING STANDARD Purchased from: USP / BP / Eupharm Analyst should document and Characterize the material Quality. In-House Prepared and Full Tested Prepared by re-crystallization of certain portion of raw Material and tested for quality

94. REFERENCE STANDARDS

95. Material selected for use as a standard should be of high purity and stability Practical requirements for reference standards differ, depending on the objective and stage in drug development cycle. REFERENCE STANDARDS Reference Standards are characterized according to the intended use as follows: (A) Primary Standards: Materials which are accepted without reference to other standards. Their identity must be provided and their purity must be high and stated (>99.0%) Characterization of primary standards generally involves Elucidation of chemical structure by tools such as; IR, UV, H-NMR, C-NMR, MS, ….. etc. Purity determination using HPLC, TLC, GC, DSC, residue of ignition, water content …..etc. Assay: Titration, DSC, Chromatography.

96. REFERENCE STANDARDS (B) Pharmacopeial Standards  Used for certain tests and assays to achieve accuracy and precision of analytical results required in the monographs.  It may be used only for the intended purpose.  Pharmacopeial standards are basically regarded as primary standards.  All standards are reference standards. (C) Working Standards  Materials are designed for daily use in analysis such as routine quality control.  They are characterized by comparison with Primary or Pharmacopoeia standards.  Their purity corresponds to a "typical batch".

97. (D) Impurity Standards:  They are mainly required for development and validation of analytical procedures (Specificity, LOD & LOQ)  For routine controls, the impurity standards are not generally needed. (E) Related compound standards: These standards are typically full characterized, but not to the same extent as primary reference standard of the active drug substance. (F) Other standards: Reagents and chemicals that are commercially available at high purity and have been characterized by trusted methods can be suitable as standard materials. REFERENCE STANDARDS

98. Handling of Standards 1. Storage:  Standard material should be protected from light, heat (2-8 0 C), and moisture (in a desiccator) and housed in a controlled environment.  Reference stock solutions and working solutions should also be labeled with the required storage conditions and expiration dates.  Containers should be sized to minimize headspace. 2.Handling  Handling must preserve integrity of the sample.  Standards that stored at sub-ambient temperatures must be equilibrated to ambient before opening to prevent water condensation on materials.  If primary standard materials are used frequently, it can be subdivided into several smaller containers to limit contamination and changes in assay.

99. REFERENCE STANDARDS 3. Documentation  A reference standard must have documentation to support its use as a standard, establish its assigned assay, and defend the retest date (or expiry date for chemical standards).  The documentation includes: (a) Reference Standard Qualification Report  A detailed qualification report is drafted for a primary reference standard of the active compound.  Less comprehensive documentation is typical for other standards.  Description of a test method used in characterization of the standard  Report results of characterization suported with data (UV, NMR spectra, HPLC fig., etc.) (b) Certificate of Analysis  All standards should have a certificate of analysis generated either from a qualified supplier or through analysis of in-house data.  The certificate should contain: Standard name, Lot number, supplier Effective data, Purity and Assay

100. Purity is freedom from foreign substances. Purity is a relative term. Absolute purity is unobtainable, and it is impossible to achieve. Reasons include the Expense, Purification Method Limitations and Stability Properties. Impurity is any material that affects the purity of the material of interest. CHEMICAL PURITY AND QUALITY CONTROL Sources of Impurities 1) Synthesis-related impurities: Originate mainly during synthetic process of raw materials, reaction vessels, reagents catalysts and solvents, intermediates and by-products. 2) Formulation-related impurities: Originate mainly from inert ingredients used to formulate the drug substance, or other reactions of drug during formulation process.

101. Thus chemicals can be classified depending on their purity to different grades include: 1) Industrial chemicals These are known as technical or commercial grade. This is the major part of chemistry production, which contains amount of impurities. 2) Pure chemicals These are reagent-grade chemicals, which have been purified as possible as could be. They are used mainly for synthesis of drugs and drug design. 3) Analytical chemicals [spectroscopic- and HPLC-grade chemicals] Are one of the most pure chemicals. Used for preparation of standard solutions, reference materials and for analytical research work. 4) Pharmaceutical grade They are very pure chemicals, which may contain traces of impurities but these impurities must be: Not harmful, not cause any undesirable effects and not contraindicated or interfere with the intended use of the pharmaceutical product. CHEMICAL PURITY AND QUALITY CONTROL

102. STABILITY The extent to which a product retains, within specified limits and throughout its period of storage & use, the same properties and characteristics that it possessed at the time of its manufacture. Factors affecting product stability 1.Stability of the active ingredient(s). 2.Potential interaction between active and inactive ingredients 3.Manufacturing process 4.Dosage form 5.Container-liner-closure system and environmental conditions encountered during shipment. 6.Handling, storage conditions and length of time between manufacture and usage. PHARMACEUTICAL PRODUCT STABILITY

103. Pharmaceutical product stability are separated into: 1.Physical stability of formulations. 2.Chemical stability of formulations. There is no absolute division between the two classes. Physical factors such as heat, light and moisture may initiate or accelerate chemical reactions, while every time a measurement is made on a chemical property, physical dimensions are included in the study. 1. Physical stability of pharmaceutical formulation Importance: (a) Pharmaceutical product must appear fresh, elegant and professional so long as it remains on the shelf. Any change such as color fading can cause patient to lose confidence in the product. PHARMACEUTICAL PRODUCT STABILITY

104. (b) Since some products are dispensed in multiple-dose containers, uniformity of dose content of active ingredient over time must be assured. (c) Active ingredient must be available to the patient throughout the expected shelf life of preparation. Physical changes in pharmaceutical products depend upon the type of dosage form itself, examples of such changes are; 1.Suspensions; Increase in crystal form of suspended active ingredient with time. 2.Solutions and emulsions; Formation of cloudy solutions and broken emulsions in addition to change of color and odor. 3.Tablets; Original size, shape, weight and color under normal handling and storage conditions as well as bioavailability of the active ingredient may be changed. 4.Gelatin capsules; Capsule shells may soften and stick together or harden with time.

105. The following factors usually cause loss of active drug content, 1. Incompatibility: Undesirable reactions between two or more components are said to result in: 1.Physical incompatibility (visibly recognizable change e.g., a gross precipitate or color change). 2.Chemical incompatibility (changes in chemical properties accompanied with visible or non visible changes) 3.Therapeutic incompatibility (includes increase of a therapeutic effect, destruction of effectiveness and occurrence of toxic manifestations).  Examples: 1- Inactivation of aminoglycoside antibiotic (Gentamycin) by pencillin in admixtures for injection due to Schiff’s base formation. Chemical stability of drug products

107. 2- Sodium metabisulfite (NaHSO 3 ) is incompatible with catechol- amines in solutions for injection: 3- Codeine is acetylated when admixed with aspirin in tablets. Chemical stability of drug products

108. 2. Racemization: It is the process of changing from an optically active compound (dextro or levo isomer) into a racemic (optically inactive or less active) mixture. Example: L-epinephrine is 15-20 times more active than its d-form while the activity of the racemic mixture is just ½ that of the L-form. In general, racemization depends on temperature, solvent, catalyst and presence or absence of light. Chemical stability of drug products

109. 3. Epimerization Ex. Tetracyclines This reaction occurs rapidly when the dissolved drug is exposed to a pH higher than 3, → steric rearrangement of the dimethylamino group. The epimer of tetracycline has little or no antibacterial activity. Chemical stability of drug products

110. 4. Hydrolysis Esters, amides and  -lactams are the compounds that are most likely to hydrolyze in presence of water. Examples: 1- Acetyl ester in Aspirin is hydrolyzed to acetic acid and salicylic acid in the presence of moisture, but in a dry environment the hydrolysis of aspirin is negligible. 2- Amide bond also hydrolyzes (e.g. chloramphenicol) but generally at a slower rate than comparable esters due its stability. Chemical stability of drug products

111. 3- Lactam and azomethine (or imine) bonds in benzodiazepines are labile to hydrolysis. Chemical stability of drug products

112. 5. Oxidation  Where addition of oxygen or removal of hydrogen is involved.  If molecular oxygen is involved, the reaction is known as auto- oxidation (occur spontaneously, slowly, at room temperature such as oxidation of Fe (II) to Fe (III) in pharmaceuticals).  e.g., phenol derivatives such as catecholamines and morphine, conjugated dienes (e.g.vitamin A and unsaturated free fatty acids), heterocyclic aromatic rings, nitrite derivatives, and aldehydes.  Products of oxidation usually lack therapeutic activity. Aldehydes (Vanillin) Catecholes (Adrenaline) Enediols (Ascorbic acid) Thiols (Captopril) Chemical stability of drug products

113. Thioethers (Promazine) Nitrites (Niclosamide) Oxidation is catalyzed by pH, polyvalent heavy metal ions (e.g., Cu(II) & Fe(III)), and UV radiations. Oxidation can be controlled by use of antioxidant chemicals, nitrogen atmospheres during ampoule and vial filling, opaque external packaging, and transparent containers. Chemical stability of drug products

114. 6. Decarboxylation It is not very common in pharmaceutical products. p-Aminosalicylic acid, Naproxen and fluoroquinolones undergo pyrolytic degradation to the decarboxylated drug and carbon dioxide. It is highly pH dependent and catalyzed by hydrogen ions and temperature. Chemical stability of drug products

115. 7. Dehydration Acid-catalyzed dehydration of tetracycline forms epianhydro-tetracycline, a product that both lacks antibacterial activity and causes toxicity. 8. Photochemical decomposition A drug can be affected chemically if it absorbs radiation of a particular wavelength. Ultraviolet radiation is the cause of many degradation reactions. The intensity and wavelength of the light and the size, shape, composition and color of the container may affect the velocity of suchreactions Example: 1- Photodegradation of Indomethacin: Chemical stability of drug products

116. 9. Ultrasonic energy and ionizing radiation  Ultrasonic energy consists of vibrations with frequency greater than 20,000 Hz,  It may cause formation of free radicals and alter drug molecules.  Example;  Changes in prednisone acetate and deoxycorticosterone acetate suspensions in an ultrasonic field have been observed.  From other hand, ionizing radiation, particularly gamma rays, which used for the sterilization of certain pharmaceutical products may cause a chemical degradation. Chemical stability of drug products

117. 10. Ionic strength In general, the hydrolysis rate constant is inversely proportional to the ionic strength with oppositely-charged ions (e.g., drug cation and excipient anions) and directly proportional to the ionic strength with ions of similar charges. 11. pH effect 1.Degradation of many drugs in solution accelerates or decelerates as the pH is decreased or increased over a specific range of pH values. 2.Improper pH ranks may cause a clinically significant loss of drug activity resulting from hydrolysis and/or oxidation reactions. 3.For example, A drug solution or suspension, may be stable for weeks, or even years in its original formulation, but when mixed with another liquid that changes pH, it degrades in minutes or days. Chemical stability of drug products

118. 12. Inter-ionic compatibility Compatibility or solubility of oppositely charged ions depends mainly on the number of charges per ion and molecular size of ions. In general, polyvalent ions of opposite charge are more likely to be incompatible. 13. Temperature  Rate of a chemical reaction increases exponentially for each 10° increase in temperature  This relationship has been observed for nearly all drug hydrolysis and some drug oxidation reactions  Suitable storage condition is a way of limiting such decompositions. Examples of such storing conditions: - Use of darkened glass - displacement of air with nitrogen Chemical stability of drug products

119. - Use of sealed containers. - Temperature controlling - Including of antioxidant e.g., ascorbic acid. The following table shows the interpretation for storage temperature instructions; Storage condition Euro Pham USP Deep-freeze -15 to 0 -20 to –10 Refrigerator 2 to 8 2 to 8 Cold >8 Cool 8 to 15 8 to 15 Room temp 15 to 25 Ambient Cont room temp 15 to 30 Chemical stability of drug products

120. What are Stability Indicating Analytical Methods (SIAMs)? They are validated quantitative test methods that can detect changes with time in the chemical, physical, or microbiological properties of drug substances or drug products. They are specific so that the quantity of the active ingredient, degradation products may be accurately measured without any interference. The ability to differentiate the active ingredient from closely related compounds and degradation products is usually the single most important requirement for stability indicating methodology. STABILITY INDICATING ASSAYS

121. When are SIAMs necessary?  To document drug substance or drug product stability.  To support a regulatory submission such as an Investigational New Drug Application (IND), Drug Master File (DMF) or to satisfy cGMP requirements.  To confirm or extend retest intervals or expiration date for active ingredients and drug products. STABILITY INDICATING ASSAYS

122. SIAMs would be achieved via three different approaches: (A) Development of highly selective method for the intact drug The degradation products are not interfere at all Examples 1- Ferric hydroxamate method for  -lactam antibiotics: Selective for penicillins and cephalosporins, Depends on the formation of colored ferric chelate with hydroxamic acid derivatives in acid medium. 2- Acid-base titration of p-aminosalicylic acid (PAS) PAS contain m- aminophenol as a major degradation product can be titrated savely against NaOH which determine only the carboxylic content STABILITY INDICATING ASSAYS

123. 3- Determination of intact drugs using difference spectrophotometry or derivative spectroscopy: Very useful in case of determination of certain drugs in presence of their oxidation or degradation products. Ex; Chlorpromazine in presence its sulphoxide and alimemazine in presence of its sulphone. 4- Spectrofluorimetric methods: More selective and sensitive than UV-methods due to the presence of two wavelegths ( exc & em ). S N CH 2 CH 2 CH 2 N(CH 3 ) 2 Cl S N CH 2 CH 2 CH 2 N(CH 3 ) 2 Cl O Chlorpromazine Chlorpromazine sulphoxide absorbed at 272 nm Absorbed at 305 nm STABILITY INDICATING ASSAYS

124. (B) Development of highly selective methods for the assay of the degradation product: Where the intact drug is determined via its degradation. Example: Aspirin (Acetyl salicylic acid) → salicylic acid + acetic acid. Salicylic acid in a degraded sample can be determined via complexation with Fe(III) to give a violet color equivalent to amount of salicylic acid present in the degraded sample STABILITY INDICATING ASSAYS

125. (C) Simultaneous separation and quanitification of both intact drug and degradation products by separation techniques  GC, HPLC, HPTLC/Densitometry and CE methods are the most common techniques used for separation and quanification STABILITY INDICATING ASSAYS