Analytical Aspect of Quality Control and Quality assurance

For pharmacy studentsBy

Dr. Abdalla Ahmed El shanawanyProfessor of Medicinal ChemistryVice Dean of Faculty of Pharmacy

Zagazig University

2010

Chapter 1: Chapter 1: Drug registrationDrug registration and and assessmentassessment

1.1. Summary of particulars1.1. Summary of particulars 1.2. Chemical and pharmaceutical 1.2. Chemical and pharmaceutical

documentationdocumentation 1.3. Reports of Experimental and 1.3. Reports of Experimental and

biological studies biological studies 1.4. Report on Clinical Studies1.4. Report on Clinical Studies 1.5. Company profile1.5. Company profile

Chapter 2: The Analytical ProblemChapter 2: The Analytical Problem 2.1. Sampling/Sample Handling2.1. Sampling/Sample Handling 2.2. Experimental errors2.2. Experimental errors 2.3. Choice of methods of an analysis 2.3. Choice of methods of an analysis 2.4. Statistics of data analysis2.4. Statistics of data analysis 2.5. Validation of analytical procedure 2.5. Validation of analytical procedure

Chapter 3Chapter 3: Drug stability and degradation product: Drug stability and degradation product 3.1. Chemical purity and its control3.1. Chemical purity and its control 3.2. International pharmacopoeia and official monograph 3.2. International pharmacopoeia and official monograph

3.3. Radiopharmaceutical3.3. Radiopharmaceutical 3.4. Stability indicating assay3.4. Stability indicating assay

Chapter 4: Function group analysisChapter 4: Function group analysis 4.1. 4.1. Classical analysisClassical analysis 4.2. Instrumental analysis4.2. Instrumental analysis 4.2.1. Spectral methods4.2.1. Spectral methods 4.2.2. Electro analytical methods4.2.2. Electro analytical methods

Chapter 5: Automation in pharmaceutical analysisChapter 5: Automation in pharmaceutical analysis 5.1. Flow injection analysis5.1. Flow injection analysis 5.2. HPLC5.2. HPLC 5.3. Gas chromatography.5.3. Gas chromatography. 5.4. Mass spectroscopy5.4. Mass spectroscopy

Chapter 6: Determination of active ingredients in Chapter 6: Determination of active ingredients in different dosage forms and in biological fluidsdifferent dosage forms and in biological fluids

6.1. Determination of active ingredients in tablets 6.1. Determination of active ingredients in tablets 6.2. Determination of active ingredients in capsules6.2. Determination of active ingredients in capsules . 6.3. Determination of active ingredients in semisolid. 6.3. Determination of active ingredients in semisolid 6.4. Determination of active ingredients in eye drops6.4. Determination of active ingredients in eye drops 6 5. Determination of active ingredients in injection6 5. Determination of active ingredients in injection 6.6. Determination of active ingredients in 6.6. Determination of active ingredients in

Suppositories.Suppositories. 6.7. Determination of active ingredients in aerosols 6.7. Determination of active ingredients in aerosols

inhalationinhalation 6.8. Radioimmunoassay6.8. Radioimmunoassay

Chapter 7: Quality assurance of pharmaceuticalsChapter 7: Quality assurance of pharmaceuticals 7.1. Good Manufacturing Practice G.M.P7.1. Good Manufacturing Practice G.M.P 7.2. National and international organization7.2. National and international organization 7.3. Departments in pharmaceutical companies7.3. Departments in pharmaceutical companies 7.4. ISO and BSI7.4. ISO and BSI

Intended Learning Outcomes of Course (lLOs)Intended Learning Outcomes of Course (lLOs)

A -Knowledge and UnderstandingA -Knowledge and Understanding Chemical QC/QA is only a part and aspect of total QC/QA Chemical QC/QA is only a part and aspect of total QC/QA

that should go hand in hand with physical and biological that should go hand in hand with physical and biological aspects and all aspects should work in perfect aspects and all aspects should work in perfect synchronization within process control, validation and other synchronization within process control, validation and other aspects of G.M.Ps.aspects of G.M.Ps.

That organization of data and documentation coupled with a That organization of data and documentation coupled with a working knowledge of relevant statistical methods is working knowledge of relevant statistical methods is mandatorymandatory

B- Intellectual SkillsB- Intellectual Skills Should be able to choose and develop suitable analytical Should be able to choose and develop suitable analytical

methodology to suit the purpose at hand so as to face various methodology to suit the purpose at hand so as to face various QC/QA settings e.g.; raw material/ single component/partially QC/QA settings e.g.; raw material/ single component/partially degraded dosage forms/ multicomponent dosage forms .degraded dosage forms/ multicomponent dosage forms .

-Given the chemical structure of the ingredients selection of -Given the chemical structure of the ingredients selection of analytical procedures should be based on a hybrid analytical procedures should be based on a hybrid methodology basis.methodology basis.

C- Professional and Practical SkillsC- Professional and Practical Skills

- To be able to apply previously known - To be able to apply previously known analytical methods to the area of drug analytical methods to the area of drug analysis analysis

D- General and Transferable SkillsD- General and Transferable Skills

- Work effectively as part of a team to - Work effectively as part of a team to collect data and/or to produce reports collect data and/or to produce reports and presentations. and presentations.

- The capability to analyze and find - The capability to analyze and find an effective solution for a given an effective solution for a given complex problem.complex problem.

Drug registrationDrug registration and and assessmentassessment

Any pharmaceutical product before it is made Any pharmaceutical product before it is made available to the market needs evaluation and available to the market needs evaluation and registration. registration.

Product licenses are required for the manufacture of Product licenses are required for the manufacture of all medicine, for sale and supply for import and all medicine, for sale and supply for import and export. Also animal test and clinical trial certificate export. Also animal test and clinical trial certificate are required.are required.

All licenses and certificates are issued by the All licenses and certificates are issued by the appropriate licensing authority which is department appropriate licensing authority which is department of health and social security, medicines division.of health and social security, medicines division.

A guideline to provide manufacturers with A guideline to provide manufacturers with information concerning documentation to be information concerning documentation to be submitted for approval and registration of submitted for approval and registration of pharmaceutical products for human use is presented.pharmaceutical products for human use is presented.

Application form for drug registrationApplication form for drug registration

1. Summary of particulars1. Summary of particulars General information relating applicant, General information relating applicant,

the license, and the productthe license, and the productdescription and name of the product, description and name of the product,

dosage form, and suggested trade dosage form, and suggested trade name physical properties of active name physical properties of active constituent recommended clinical use, constituent recommended clinical use, dose, route of administration, place of dose, route of administration, place of manufacture, sale, OTC or by manufacture, sale, OTC or by prescription and supplyprescription and supply

2. Chemical and pharmaceutical documentation2. Chemical and pharmaceutical documentation

2.1. 2.1. Chemical Data on active ingredientChemical Data on active ingredient

Nonproprietary or generic name, Nonproprietary or generic name, molecular formula, chemical name, structure, molecular formula, chemical name, structure, physicochemical properties, synthesis, physicochemical properties, synthesis, stability studies, analytical specifications and stability studies, analytical specifications and test methods, key raw materials, key test methods, key raw materials, key intermediates, degradation profile, including intermediates, degradation profile, including analytic procedures used in the detection and analytic procedures used in the detection and determination of byproducts.determination of byproducts.

2.2. 2.2. Formulation Report (dosage forms)Formulation Report (dosage forms) Data on CompositionData on Composition

Complete qualitative and quantitative Complete qualitative and quantitative composition of the finished product, including quality composition of the finished product, including quality specifications (requirements) and control methods. specifications (requirements) and control methods. Active ingredient(s) present in the form of salts or Active ingredient(s) present in the form of salts or hydrates shall be described quantitatively by their hydrates shall be described quantitatively by their total mass and by the mass of the active moiety or total mass and by the mass of the active moiety or moieties of the molecule.moieties of the molecule.

Data on Packaging Materials (Container and Data on Packaging Materials (Container and Closures)Closures)

Detailed information is required about the Detailed information is required about the packaging material, which comes in to such contact packaging material, which comes in to such contact with the drug Package labeling includes package with the drug Package labeling includes package leaflet, label on the immediate container, and outer leaflet, label on the immediate container, and outer wrapper or carton.wrapper or carton.

The package leaflet should consist of factual and The package leaflet should consist of factual and scientific information consistent with the application. A scientific information consistent with the application. A leaflet must bear adequate information for use.leaflet must bear adequate information for use.

2.3. Analytical Report2.3. Analytical Report The manufacturer should submit:The manufacturer should submit: Quality specifications (requirements) Quality specifications (requirements)

and analytical procedures for the and analytical procedures for the dosage form dosage form

2.4. Stability Report2.4. Stability Report The stability report should consist The stability report should consist

of stability data sheet and a summary. of stability data sheet and a summary. The tests for stability at each sampling The tests for stability at each sampling period should be related to the period should be related to the formulation and to the storage condition formulation and to the storage condition and the study should include tests.and the study should include tests.

3. Reports of Experimental and biological 3. Reports of Experimental and biological studies. (Pre clinical studies)studies. (Pre clinical studies)

3.1. 3.1. Animal PharmacologyAnimal Pharmacology The manufacturer should furnish a summary The manufacturer should furnish a summary

of the observations and conclusions Showing the of the observations and conclusions Showing the animal species, number of animals, doses, animal species, number of animals, doses, information route of administration, concise information route of administration, concise description of the methodology, results, description of the methodology, results, conclusions and an overall evaluation of the conclusions and an overall evaluation of the pharmacodynamic and pharmacokinetics pharmacodynamic and pharmacokinetics properties of the drug based on the findings in properties of the drug based on the findings in laboratory animals or in invitro systems.laboratory animals or in invitro systems.

3.2. Pharmacodynamics3.2. Pharmacodynamics Studies providing the primary basis for clinical Studies providing the primary basis for clinical

trials of the drug, mechanism of action, minimum trials of the drug, mechanism of action, minimum effective dose emphasizing adequate description effective dose emphasizing adequate description of dose-effect relationships that produce of dose-effect relationships that produce pharmacological responses in each species of pharmacological responses in each species of animal investigated.animal investigated.

3.3. Pharmacokinetics3.3. Pharmacokinetics Pharmacokinetics Studies concerning absorption, Pharmacokinetics Studies concerning absorption,

distribution, metabolism, enzyme induction, enzyme distribution, metabolism, enzyme induction, enzyme inhibition, and excretion.inhibition, and excretion.

3.4. Toxicological Data3.4. Toxicological Data Summary of toxicological studies preferably Summary of toxicological studies preferably

should be presented in tables which indicate species, should be presented in tables which indicate species, number, sex, age, weight and strain of animals, number, sex, age, weight and strain of animals, information on dosage formulation, route(s) of information on dosage formulation, route(s) of administration, treatment regimen duration of administration, treatment regimen duration of treatment, parameters evaluated, significant treatment, parameters evaluated, significant observations and conclusions.observations and conclusions.

3.5. Microbiology (for anti-microbial agents 3.5. Microbiology (for anti-microbial agents only)only)

Summaries of all microbiologic studies, including Summaries of all microbiologic studies, including methods used together with a discussion and methods used together with a discussion and evaluation of the results. Cumulative MICevaluation of the results. Cumulative MIC tables are tables are highly desirable.highly desirable. Under each of the following Under each of the following headings, detailed description and analysis based on headings, detailed description and analysis based on the available completed microbiologic studies should the available completed microbiologic studies should be provided.be provided.

4. Report on Clinical Studies4. Report on Clinical Studies Drug clinical trials shall be conducted in Drug clinical trials shall be conducted in

institutions legally certified for drug clinical trials. If a institutions legally certified for drug clinical trials. If a clinical trial has to be conducted by an institution not clinical trial has to be conducted by an institution not yet certified, a special approval by the State Food yet certified, a special approval by the State Food and Drug Administration should be obtained.and Drug Administration should be obtained. The investigator responsible for a drug clinical The investigator responsible for a drug clinical trial shall, in accordance with the relevant provisions, trial shall, in accordance with the relevant provisions, timely report adverse events occurred in the process timely report adverse events occurred in the process of the clinical trial to the State Food and Drug of the clinical trial to the State Food and Drug Administration. Administration.

4.1. Clinical Pharmacology4.1. Clinical Pharmacology:: PharmacodynamicsPharmacodynamics Intended drug effect, methodology, number of Intended drug effect, methodology, number of

volunteers, age groups, healthy and sick, optimal volunteers, age groups, healthy and sick, optimal dose Studies of single and multiple dose, And the dose Studies of single and multiple dose, And the effect of drugs on various organic functions, effect of drugs on various organic functions, mechanism of action, studies on the relationship of mechanism of action, studies on the relationship of between dose of drug and response in patient, drug between dose of drug and response in patient, drug interaction studies, etc.interaction studies, etc.

PharmacokineticsPharmacokinetics

Studies on the absorption, distribution plasma Studies on the absorption, distribution plasma concentration, protein binding, half- life, concentration, protein binding, half- life, biotransformation, kinetics, elimination of the drug and biotransformation, kinetics, elimination of the drug and also report on metabolic studies. Physicochemical also report on metabolic studies. Physicochemical properties which may act on absorption and distribution properties which may act on absorption and distribution should be stated. The methods of assay or determination should be stated. The methods of assay or determination should be specified. should be specified.

Reports on combined preparations, in addition to details Reports on combined preparations, in addition to details on their individual components, should also give on their individual components, should also give information on the pharmacological properties of the information on the pharmacological properties of the particular combination that is being considered.particular combination that is being considered.

4.2. 4.2. Bioavailability ReportBioavailability Report

Bioequivalence report is required for those oral Bioequivalence report is required for those oral dosage forms of drugs which are known to pose dosage forms of drugs which are known to pose bioavailability problem, where systemic absorption is a bioavailability problem, where systemic absorption is a requirement for their efficacy.requirement for their efficacy.

4.3. Clinical Trials4.3. Clinical Trials

The summary should concisely set out the clinical The summary should concisely set out the clinical properties of the drug. Special emphasis should be put on properties of the drug. Special emphasis should be put on that documentation which lends support to the cited that documentation which lends support to the cited indications. indications.

It should provide information on patient population number It should provide information on patient population number of patients, dosage formulation, doses, methods, etc; and of patients, dosage formulation, doses, methods, etc; and also give an overall discussion and evaluation of the safety, also give an overall discussion and evaluation of the safety, efficacy, dosages, adverse reactions and contraindications of efficacy, dosages, adverse reactions and contraindications of the drug based on the findings of available completed clinical the drug based on the findings of available completed clinical trials, and conclusion providing a discussion of the benefits trials, and conclusion providing a discussion of the benefits and risks of the drug under the conditions of use and risks of the drug under the conditions of use recommended.recommended.

The conclusion should at least consider the following points:The conclusion should at least consider the following points:

comparison of the expected clinical benefits with possible comparison of the expected clinical benefits with possible adverse effects, and assessment and comparison of the adverse effects, and assessment and comparison of the benefit/risk ratio of the drug in relation to related drugs or benefit/risk ratio of the drug in relation to related drugs or others used as standards in controlled clinical trials, etc. others used as standards in controlled clinical trials, etc.

5. Company profile 5. Company profile Documents to be supplied by the manufacturer.Documents to be supplied by the manufacturer.

5.1. 5.1. Back ground informationBack ground information The manufacturer should submit background information about the The manufacturer should submit background information about the

company indicating, Year of establishment, Development since company indicating, Year of establishment, Development since establishment, , Total working force, Ownership,establishment, , Total working force, Ownership,

5.2. 5.2. Production UnitProduction Unit The manufacturer should submit information on the production unit The manufacturer should submit information on the production unit

and should also indicate whether the company has the following;and should also indicate whether the company has the following; GMP procedure, Master file and batch production record systemGMP procedure, Master file and batch production record system Product specifications, Standard operation manual, List of pharmaceuticals Product specifications, Standard operation manual, List of pharmaceuticals

produced by the manufacturer (specify those which are the manufacturer's produced by the manufacturer (specify those which are the manufacturer's innovation).innovation).

5.3. Quality Control Unit5.3. Quality Control Unit The manufacturer should state:The manufacturer should state: A. Whether it performs, Raw & packaging materials Q.C, A. Whether it performs, Raw & packaging materials Q.C, In-process Q.C., and Finished product Q.C.In-process Q.C., and Finished product Q.C. B. The types of Q.C. tests performed; physicochemical tests, SterilityB. The types of Q.C. tests performed; physicochemical tests, Sterility test , Pyrogen test, acute toxicity test, Biological test , Pyrogen test, acute toxicity test, Biological assay, Microbiological assay etc.assay, Microbiological assay etc. C. Whether it has Good laboratory Practice (GLP) Procedure.C. Whether it has Good laboratory Practice (GLP) Procedure. D. The major Q.C. Instruments available.D. The major Q.C. Instruments available. E. Qualification and experience of Q.C. personnelE. Qualification and experience of Q.C. personnel

5.4. 5.4. Supply systemSupply system The manufacturer should give information on its supply The manufacturer should give information on its supply

system indicating whether it has at least the following:system indicating whether it has at least the following: Cold storage facilities, Separate stores for raw materials, Cold storage facilities, Separate stores for raw materials,

packaging materials, labels etc., Separate room for weighing raw packaging materials, labels etc., Separate room for weighing raw materials, Quarantine for raw materials, finished products, etc,, materials, Quarantine for raw materials, finished products, etc,, procedure for supplies control.procedure for supplies control.

5.5. Research and Development Unit (R and D)5.5. Research and Development Unit (R and D) The manufacturer should give detailed information on at The manufacturer should give detailed information on at

least the following major points:least the following major points: The year R and D was initiated.The year R and D was initiated. Qualification of the personnel engaged in R and D activitiesQualification of the personnel engaged in R and D activities Major research areas and achievements attained.Major research areas and achievements attained. Affiliation with other institutes (if there is any)Affiliation with other institutes (if there is any)

5.6. Product Registration and marketing Experience of the 5.6. Product Registration and marketing Experience of the manufacturermanufacturer

The manufacturer should submit full information on its The manufacturer should submit full information on its marketing experience and Registration status of its products marketing experience and Registration status of its products indicating:indicating:

List of countries to which it exports most of its products.List of countries to which it exports most of its products. List of countries in which its products are registeredList of countries in which its products are registered

Chapter 2: Analytical problem Chapter 2: Analytical problem

2.1 Sampling/Sample Handling2.1 Sampling/Sample Handling

2.2 Experimental errors2.2 Experimental errors

2.3 Choice of methods of an analysis2.3 Choice of methods of an analysis

2.4 Statistics of data analysis2.4 Statistics of data analysis

2.5 Validation Of analytical procedure.2.5 Validation Of analytical procedure.

There are three basic activities involved There are three basic activities involved in solving an analytical problemin solving an analytical problem::

1. Collection of the applicable sample1. Collection of the applicable sample

2. Preparation of the sample for analysis2. Preparation of the sample for analysis

3. Analysis using appropriate methods.3. Analysis using appropriate methods.

2.1 Sampling/Sample Handling2.1 Sampling/Sample Handling Sample is a representative portion selected from the Sample is a representative portion selected from the

bulk.bulk. One of the most common causes of differing analytical results One of the most common causes of differing analytical results

can be traced back to non representative ness of different can be traced back to non representative ness of different samples.samples.

It is easy to anticipate that this could occur during the conduct It is easy to anticipate that this could occur during the conduct of a multi-lab study, and it is the responsibility of the study of a multi-lab study, and it is the responsibility of the study director to ensure sample homogeneity. director to ensure sample homogeneity.

When a significant difference in results occurs between When a significant difference in results occurs between laboratories that have analyzed supposedly the same sample, a laboratories that have analyzed supposedly the same sample, a serious problem may arise Involving questions of competence serious problem may arise Involving questions of competence and credibility. Many of theseand credibility. Many of these situations can be avoided if situations can be avoided if samples are collected according to a rationalsamples are collected according to a rational plan that gives plan that gives some assurance that the sample delivered to thesome assurance that the sample delivered to the laboratory laboratory represents the composition of the parent lot.represents the composition of the parent lot.

For example when the U.S. Environmental Protection For example when the U.S. Environmental Protection Agency conducted studies during remediation projects to Agency conducted studies during remediation projects to determine the sources of variation in the sampling and determine the sources of variation in the sampling and analytical procedures, and found that the amount of variation analytical procedures, and found that the amount of variation from sampling was approximately 80% of the total variation from sampling was approximately 80% of the total variation while the amount of variation from the analytical procedures while the amount of variation from the analytical procedures was 20%. The important point in this example is to show that was 20%. The important point in this example is to show that sampling error can play a very significant part in the overall sampling error can play a very significant part in the overall error in the analytical system.error in the analytical system.

Analytical sampleAnalytical sample Analytical sample is a small portion selected Analytical sample is a small portion selected

from the sample.from the sample. The best analysis can give misleading information The best analysis can give misleading information

if the test portion analyzed does not represent the if the test portion analyzed does not represent the sample or the lot from which it was taken. sample or the lot from which it was taken. Distortions introduced at this point will carry Distortions introduced at this point will carry through the analysis and adversely affect the final through the analysis and adversely affect the final results and the conclusions drawn from them.results and the conclusions drawn from them.

A homogenous materialA homogenous material, such as a liquid , such as a liquid solution, readily yields a representative sample.solution, readily yields a representative sample.

Heterogeneous materialsHeterogeneous materials, such as powdered , such as powdered materials and granules always exhibit segregation materials and granules always exhibit segregation of the particles during handling .and exhibit a of the particles during handling .and exhibit a range of particle size, and the flow properties of range of particle size, and the flow properties of the powder depend upon the particle size the powder depend upon the particle size distribution distribution

Two techniques for obtaining good Two techniques for obtaining good

sample from powderedsample from powdered materialmaterial:: Long pile methodLong pile method The gross sample is The gross sample is

arranged in long pile, and then separated arranged in long pile, and then separated into two equal piles using a shovel and into two equal piles using a shovel and throwing alternative shovels to opposite throwing alternative shovels to opposite sides. One half is discarded and the other sides. One half is discarded and the other again separated in the same manner until again separated in the same manner until the required sample size is obtained. the required sample size is obtained.

Long pile2.2. Coning and Coning and quarteringquartering

The powder is deposited on a flat The powder is deposited on a flat surface by shovel, and then the cone is surface by shovel, and then the cone is flattened and divided into four equal parts flattened and divided into four equal parts by forming two perpendicular diameters. by forming two perpendicular diameters. The material from two diagonally opposed The material from two diagonally opposed quarters is combined and formed into quarters is combined and formed into another cone, that material in the other another cone, that material in the other two quarters being set aside .the coning two quarters being set aside .the coning and quartering process is continued until and quartering process is continued until a sample of the requisite size is obtained.a sample of the requisite size is obtained.

coning and qartering

When the populations consist of When the populations consist of discrete units, such discrete units, such as drums of solvent, package of ampoules, as drums of solvent, package of ampoules, bottles, etc., there are two general sampling bottles, etc., there are two general sampling approachesapproaches

Random samplingRandom sampling The units are numbered serially .all numbered have The units are numbered serially .all numbered have

an equal number of digits. Then numbers are selected in an equal number of digits. Then numbers are selected in some random manner from the random table, and the some random manner from the random table, and the corresponding units are taken for the sample. It is an corresponding units are taken for the sample. It is an effective way to obtain unbiased, representative effective way to obtain unbiased, representative samples, but it is apt to be laborious and time samples, but it is apt to be laborious and time consuming.consuming.

Systematic samplingSystematic sampling A more widely used time saving technique in which A more widely used time saving technique in which

every nth unit is selected to constitute the sample. The every nth unit is selected to constitute the sample. The selection of n is of course critical, as the interval selection of n is of course critical, as the interval between selected units must not correspond to any between selected units must not correspond to any periodicity in the population. It is biased to some extent.periodicity in the population. It is biased to some extent.

Sample size

The n plan should be used only when the bulk is homogenous and the sample can be withdrawn from any part of the container n= √N

where n is the sample size, N is the quantity to be sampled

The p plan For homogenous and the sample for identity P= 0.4√ N

The r plan For heterogonous and vegetable drug as raw materials r= 1.5√ N, where r is the sample size. N is the quantity to be sampled

There are generally two choices specify the manner in which an analytical sample should be taken.

1. Preparation of a composite laboratory sample (if multiple units are submitted for analysis)

2. Examination of individual units. A composite laboratory sample is one in which the individual units, or

Representative portions of units are mixed to form a uniform mixture. Portions are then taken from the composite for analysis

Compositing can Best be used when homogeneity is not a significant problem concern.

Compositing saves analytical time and in some types of contract testing it May be the procedure specified.

Compositing is not the procedure of choice when there is a chance that an individual unit that constitutes a public health or safety threat will not be detected (there are some exceptions) or where a unit at or outside tolerance will not be detected because of matrix dilution.

Multiple unit laboratory sampling is indicated when the possible range of Values among individual units are considered significant or it is desirable to establish the variability of the lot.

However, the reliability of the result generally increases with the square root of the number of samples analyzed. For this reason, analyses of multiple samples always are preferred over single samples since single samples give no information on the homogeneity of the lot that was samples

Sample Preparation for AnalysisSample Preparation for Analysis

Every type of material that is to be prepared for analysis Every type of material that is to be prepared for analysis presents its own practical difficulties. The requirements for presents its own practical difficulties. The requirements for suitable suitable sample preparation are dictated by the consistency and thesample preparation are dictated by the consistency and the chemical chemical characteristics of the analyte and characteristics of the analyte and the matrixthe matrix, and by the , and by the distribution distribution of the analyteof the analyte in the sample. Even seemingly homogeneous in the sample. Even seemingly homogeneous materials such as liquids may be subject to Sedimentation or materials such as liquids may be subject to Sedimentation or stratification.stratification.

Single phase liquids can generally be mixed, stirred, shaken,Single phase liquids can generally be mixed, stirred, shaken, Dry particulate materials can be reduced in volume by use of a Dry particulate materials can be reduced in volume by use of a

splitter, A variety of implements and machines are available for splitter, A variety of implements and machines are available for sample disintegration, such as mills, grinders and cutters. Care in sample disintegration, such as mills, grinders and cutters. Care in their use is necessary to prevent loss of dust or change in their use is necessary to prevent loss of dust or change in composition through the partial separation of components. In composition through the partial separation of components. In particular, care must be taken to prevent dust or related substances particular, care must be taken to prevent dust or related substances as carry over, from contaminating the laboratory space and any as carry over, from contaminating the laboratory space and any subsequent samples that are ground, and of course, the grinding subsequent samples that are ground, and of course, the grinding equipment must be meticulously cleaned between samples. equipment must be meticulously cleaned between samples. Screening can be used to improve the efficiency of particle size Screening can be used to improve the efficiency of particle size reduction, followed by mixing to attain homogeneity.reduction, followed by mixing to attain homogeneity.

Glass containers and laboratory apparatus can adsorb Glass containers and laboratory apparatus can adsorb certain materials and may require surface treatment.certain materials and may require surface treatment.

Plastic containers can retain contaminants, such as Plastic containers can retain contaminants, such as animal hairs, while the rest of the sample is animal hairs, while the rest of the sample is transferred with apparent ease. In other words, transferred with apparent ease. In other words, validation of a method of analysis includes, most validation of a method of analysis includes, most certainly, validation of the method of sample certainly, validation of the method of sample preparation and storage.preparation and storage.

Loss or gain of moisture during manipulation can be a Loss or gain of moisture during manipulation can be a problem. Loss can be minimized by keeping samples problem. Loss can be minimized by keeping samples covered with plastic or aluminum foil. A cold product covered with plastic or aluminum foil. A cold product can be protected from gaining moisture by allowing can be protected from gaining moisture by allowing the sample to come to room temperature before the sample to come to room temperature before preparation begins.preparation begins.

When volatile organic constituents are present in When volatile organic constituents are present in samples, sample manipulation may not be possible, or samples, sample manipulation may not be possible, or may be severely restricted, in order to prevent their may be severely restricted, in order to prevent their loss.loss.

Experimental Errors Experimental Errors Random Random (in determinant)(in determinant) and systematic and systematic (determinant)(determinant)

errorserrors The term error as used here refers to the numerical The term error as used here refers to the numerical

difference between a measured value and the true value.difference between a measured value and the true value. For example, the % composition of a standard sample For example, the % composition of a standard sample

certified by the national Bureau of standards may be treated certified by the national Bureau of standards may be treated as correct in evaluating a new analytical method; differences as correct in evaluating a new analytical method; differences between the standard values and the results obtained by the between the standard values and the results obtained by the new method are then treated as error in the latter. new method are then treated as error in the latter.

One can attempt to One can attempt to minimizeminimize errors but cannot eliminate errors but cannot eliminate them completelythem completely

Random errors Random errors small fluctuations introduced in nearly all analyses. Arise small fluctuations introduced in nearly all analyses. Arise

from variation of external conditions over which the observer from variation of external conditions over which the observer has no control For example, repeated measurements of the has no control For example, repeated measurements of the same property often differ even if they are performed on a same property often differ even if they are performed on a single instrument that is calibrated and operated properly. single instrument that is calibrated and operated properly.

Systematic errors Systematic errors Systematic errors cause the results to vary from the correct value in Systematic errors cause the results to vary from the correct value in

a predictable manner and can often be identified and corrected.a predictable manner and can often be identified and corrected. An example of a systematic error is improper calibration of an An example of a systematic error is improper calibration of an

instrument. The measurement lacks instrument. The measurement lacks accuracyaccuracy. It is even possible that . It is even possible that repeated measurements with this broken instrument will give repeated measurements with this broken instrument will give reproducible results (high precision), but every one of them will deviate reproducible results (high precision), but every one of them will deviate from the true value (low accuracy). from the true value (low accuracy).

Systematic errorsSystematic errors have been classified as personal, instrumental and have been classified as personal, instrumental and methodicmethodic

1. 1. personal errorspersonal errors:: Are those due to the carelessness of the observer and include such Are those due to the carelessness of the observer and include such

things as misreading a burette or overshooting the end point, or loss in things as misreading a burette or overshooting the end point, or loss in weight during washing and filtration, and mathematic error in weight during washing and filtration, and mathematic error in calculations.calculations.

2. 2. Instrumental error:Instrumental error: Error due to the instruments themselves and include the use of Error due to the instruments themselves and include the use of

uncalibrated equipments e.g. Weights, burettes, pipette, flasks.uncalibrated equipments e.g. Weights, burettes, pipette, flasks.

3. 3. Methodic: Methodic: Errors are those inherent in the method itself, incomplete reaction Errors are those inherent in the method itself, incomplete reaction

or side reaction, instability of the reaction product or impurities in the or side reaction, instability of the reaction product or impurities in the reagents.reagents.

Minimization of the errorsMinimization of the errors

1-Calibration of the equipment and application of correction 1-Calibration of the equipment and application of correction factorfactor..

All of the equipments should be corrected and appropriate All of the equipments should be corrected and appropriate correction factor applied.correction factor applied.

2-Use of blank experiments2-Use of blank experiments Carrying out a separate determination without the sample Carrying out a separate determination without the sample

under the same experimental condition to overcome the effect of under the same experimental condition to overcome the effect of impurities introduced through reagents, solvents and vessels.impurities introduced through reagents, solvents and vessels.

3. 3. Standard addition methodStandard addition method A known amount of the constituent being determined (standard) is A known amount of the constituent being determined (standard) is

added to the sample which is then analyzed for the total amount added to the sample which is then analyzed for the total amount of the constituent present. The difference between the analytical of the constituent present. The difference between the analytical results for the sample with and without the added standard gives results for the sample with and without the added standard gives the recovery of the amount of added constituent.the recovery of the amount of added constituent.

4. 4. Use of internal standardUse of internal standard It is used in HPLC and GC; fixed amount of the suitable It is used in HPLC and GC; fixed amount of the suitable

standard is added with different amount of the sample to minimize standard is added with different amount of the sample to minimize the error during introduction of the sample. the error during introduction of the sample.

Choice of methods of an analysisChoice of methods of an analysis

The pharmacist who has need for analytical data usually finds himself The pharmacist who has need for analytical data usually finds himself faced with an array of methods which could be used to provide the desired faced with an array of methods which could be used to provide the desired information. Unfortunately, there are no generally applicable rules that can information. Unfortunately, there are no generally applicable rules that can be applied; the choice of method is thus a matter of judgment. Such be applied; the choice of method is thus a matter of judgment. Such judgment is difficult, and the ability to make it will come only with judgment is difficult, and the ability to make it will come only with experience.experience.

Factors Factors plays an important role in the selection of the analytical plays an important role in the selection of the analytical methodsmethods of analysis. of analysis.

1-Concentration range of the sample to be determined.1-Concentration range of the sample to be determined. if for example the element present to the extent of a few ppm as if for example the element present to the extent of a few ppm as

impurities or degradation products it is not suitable to use volumetric or impurities or degradation products it is not suitable to use volumetric or gravimetric methods and use other gravimetric methods and use other sensitive sensitive methods as HPLC or methods as HPLC or spectrophotometry. On other hand if the analyte is a major component of spectrophotometry. On other hand if the analyte is a major component of the sample the classical method may be preferable.the sample the classical method may be preferable.

2-Degree of accuracy and precision required 2-Degree of accuracy and precision required The accuracy and precision required are of vital importance in the The accuracy and precision required are of vital importance in the

choice of an analytical method and its performance, especially in small and choice of an analytical method and its performance, especially in small and vital substances as hormones.vital substances as hormones.

3- Different components which are present in the sample3- Different components which are present in the sample The chemical structure of these substances should be known because The chemical structure of these substances should be known because

they may interfere with the method, since the analysis is based on they may interfere with the method, since the analysis is based on reactions or properties share by several compounds, in this case prior reactions or properties share by several compounds, in this case prior separation or selective method should be used.separation or selective method should be used.

4-Physical and chemical properties of the substance4-Physical and chemical properties of the substance The analyst should know the state of the substance at The analyst should know the state of the substance at

ordinary conditions and whether losses by volatility, and ordinary conditions and whether losses by volatility, and whether or not the sample is hygroscopic or efflorescent whether or not the sample is hygroscopic or efflorescent and what sort of treatment is sufficient to decompose or and what sort of treatment is sufficient to decompose or dissolve the sample without loss of the analyte.dissolve the sample without loss of the analyte.

5-Number of the sample to be analyzed5-Number of the sample to be analyzed If there are many considerable times can be expended in If there are many considerable times can be expended in

calibrating instruments, preparing reagents, assembling calibrating instruments, preparing reagents, assembling equipments, the cost of these operations can be spread equipments, the cost of these operations can be spread over the large number of analyses.over the large number of analyses.

On other hand, if a few samples are to be analyzed, a On other hand, if a few samples are to be analyzed, a longer and more tedious procedure involving a minimum of longer and more tedious procedure involving a minimum of these preparatory operations may actually prove to be the these preparatory operations may actually prove to be the wiser choice from the economic standpoint.wiser choice from the economic standpoint.

6-Availability of the instruments and equipments6-Availability of the instruments and equipments.. plays an important role in the selection of the analytical plays an important role in the selection of the analytical

method.method.

Testing the procedureTesting the procedure

Once a procedure for an analysis has been selected, the Once a procedure for an analysis has been selected, the problem usually arises as to whether the method can be employed problem usually arises as to whether the method can be employed directly without testing. The answer depends upon a number of directly without testing. The answer depends upon a number of considerations. If the procedure chosen has been the subject of a considerations. If the procedure chosen has been the subject of a single or a few literature references Or a major modification of a single or a few literature references Or a major modification of a standard procedure is undertaken Or an attempt is made to apply standard procedure is undertaken Or an attempt is made to apply it to a type of sample different from that for which it was it to a type of sample different from that for which it was designed; a preliminary laboratory test is advisabledesigned; a preliminary laboratory test is advisable

What are the means by which a new method or a modification of What are the means by which a new method or a modification of an existing method can be tested for reliability?an existing method can be tested for reliability?

1-Analysis of standard sample1-Analysis of standard sample

The best technique for evaluating an analytical method The best technique for evaluating an analytical method involves the analysis of one or more standard samples whose involves the analysis of one or more standard samples whose composition with respect to the compound of interest is reliably composition with respect to the compound of interest is reliably known.known.

For this technique to be of value, it is essential that the standards For this technique to be of value, it is essential that the standards closely resemble the sample to be analyzed with respect to both closely resemble the sample to be analyzed with respect to both the concentration range of the analyte and the overall the concentration range of the analyte and the overall composition. Standard may be purchased from sources such as composition. Standard may be purchased from sources such as national Bureau of standards. Or synthesized from weighed national Bureau of standards. Or synthesized from weighed quantities of pure compounds.quantities of pure compounds.

Analysis by other methodsAnalysis by other methods

The result of an analytical method can sometimes be The result of an analytical method can sometimes be evaluated by comparison with some entirely different evaluated by comparison with some entirely different method. It should be based on chemical principles that method. It should be based on chemical principles that differ considerably from that one under examination. differ considerably from that one under examination. Comparable result from the two methods, serve as Comparable result from the two methods, serve as presumptive evidence that both are yielding satisfactory presumptive evidence that both are yielding satisfactory results; no significant difference between the two methods.results; no significant difference between the two methods.

Standard addition to the sampleStandard addition to the sample

Standard addition method, in addition to being used Standard addition method, in addition to being used to analyze the sample itself, the proposed procedure is to analyze the sample itself, the proposed procedure is tested against portions of the sample to which known tested against portions of the sample to which known amounts of the analyte have been added.amounts of the analyte have been added.

The effectiveness of the method can be established by The effectiveness of the method can be established by evaluating the extent of recovery of the added quantity.evaluating the extent of recovery of the added quantity.

The standard addition method may reveal errors arising The standard addition method may reveal errors arising from the method of treating the sample or from the from the method of treating the sample or from the presence of the other compoundspresence of the other compounds

Rejection of DataRejection of Data

Sometimes a person performing measurements is faced with Sometimes a person performing measurements is faced with one result in a set of replicate s which seems to be out of line one result in a set of replicate s which seems to be out of line with others, and he then must decide whether to exclude this with others, and he then must decide whether to exclude this result from further consideration.result from further consideration.

For example, after making only five measurements of our For example, after making only five measurements of our

drug, you obtain the following results (in grams):drug, you obtain the following results (in grams): 8.148 8.145 8.156 8.149 8.1778.148 8.145 8.156 8.149 8.177 The last measurement seems a bit off, and you may be The last measurement seems a bit off, and you may be

tempted to throw it out of the set. However, you must never tempted to throw it out of the set. However, you must never throw out a result from a data set unless you have a throw out a result from a data set unless you have a statistical reason to do so.statistical reason to do so.

Statistically speaking, we are asking the question: Statistically speaking, we are asking the question: does the measurement of 8.177 grams belong to the same does the measurement of 8.177 grams belong to the same normal distribution as the other four measurements?normal distribution as the other four measurements?

. If we had a very large. If we had a very large data set data set, then we could calculate x ± , then we could calculate x ± 2sx , and then determine if the measurement in question 2sx , and then determine if the measurement in question falls outside the confidence interval. However, our data set is falls outside the confidence interval. However, our data set is very small (N < 10), so that the standard deviation alone is very small (N < 10), so that the standard deviation alone is not a good criterion for rejection.not a good criterion for rejection.

Statisticians have devised many rejection tests for Statisticians have devised many rejection tests for the detection of non-random errors. We will describe only the detection of non-random errors. We will describe only one - the Q test - which works well in cases where 3 < N < one - the Q test - which works well in cases where 3 < N < 10.10.

In order to test the value of 8.177 grams, we must calculate the so-called In order to test the value of 8.177 grams, we must calculate the so-called Qcalc value for this observation. Qcalc value for this observation.

Q calc Q calc absolute value of the gap between the suspect value and the value absolute value of the gap between the suspect value and the value closest to it / range of values closest to it / range of values

To calculate Qcalc in our example, we display the data in increasing order of To calculate Qcalc in our example, we display the data in increasing order of numerical value, and then identify the suspect value and the value that is numerical value, and then identify the suspect value and the value that is closest to it:closest to it:

8.145 8.148 8.149 8.145 8.148 8.149 8.156 8.156 8.177 8.177 Then,Then, Q calc = 8 .177 - 8.156 / 8. 177 - 8 .145 = 0 .66Q calc = 8 .177 - 8.156 / 8. 177 - 8 .145 = 0 .66 We now compare this Q calc with a We now compare this Q calc with a critical value critical value Q c. If Q calc > Q c, then Q c. If Q calc > Q c, then

the observation may be rejected. If Q calc < Q c, then we must keep the the observation may be rejected. If Q calc < Q c, then we must keep the observation. observation.

The Qc value depends on the confidence level and the number of The Qc value depends on the confidence level and the number of observations in your set. A partial list follows:observations in your set. A partial list follows:

N Q c (90% confidence) N Q c (90% confidence) 3 0.943 0.94 4 0.764 0.76 5 0.645 0.64 6 0.566 0.56 7 0.517 0.51 8 0.478 0.47 9 0.449 0.44 10 0.4110 0.41 Returning to our example, where N = 5, we see that Q calc = 0.66 is Returning to our example, where N = 5, we see that Q calc = 0.66 is

indeed greater than QC = 0.64. Hence, we are justified in rejecting the indeed greater than QC = 0.64. Hence, we are justified in rejecting the observation. However, you must indicate in your report that the Q test was observation. However, you must indicate in your report that the Q test was used at a 90 % confidence interval.used at a 90 % confidence interval.

Testing for significanceTesting for significance

Suppose that a sample is analyzed by two different methods, Suppose that a sample is analyzed by two different methods, each repeated several times and the mean values obtained are each repeated several times and the mean values obtained are different.different.

Is the difference between the two values significant?Is the difference between the two values significant? We use null hypothesis which states that the two means are We use null hypothesis which states that the two means are

identical and the student, s t test gives a yes or no answer to the identical and the student, s t test gives a yes or no answer to the correctness of the null hypothesis with a certain confidence.correctness of the null hypothesis with a certain confidence.

Procedure:Procedure: Suppose a sample has been analyzed by two different methods; Suppose a sample has been analyzed by two different methods;

yielding means Xyielding means X--11 ,and x- ,and x- 2 2 and standard deviation s and standard deviation s1 1 and s and s22 ; n ; n11, ,

and nand n2 2 are the number of the individual results obtained by the two are the number of the individual results obtained by the two methods.methods.

First calculate tFirst calculate t xx11- - x-- - x-22 t =------------------t =------------------ Sp√ 1/nSp√ 1/n11 + 1/n + 1/n22 Where Where (n(n11-1) s-1) s11

22 + (n + (n22 -1) s -1) s2222

Sp (pooled sd) = √ ------------------------Sp (pooled sd) = √ ------------------------ nn1 1 +n+n22 -2 -2 sec., find t tabulated at degree of freedom nsec., find t tabulated at degree of freedom n11+n+n22 -2 and at the -2 and at the

desired probability if the t value in the table less than the desired probability if the t value in the table less than the calculated t , the null hypothesis incorrect and there is significant calculated t , the null hypothesis incorrect and there is significant difference between the two means. difference between the two means.

F test (variance ratio)F test (variance ratio) To decide whether the difference between sTo decide whether the difference between s11 and s and s22 is is

significantsignificant F = SF = S11

22/ S/ S2222 F > 1 F > 1

The larger s in the numeratorThe larger s in the numerator

ExampleExample Aspirin sample was analyzed by two different methods and Aspirin sample was analyzed by two different methods and

the following results were giventhe following results were given Method 2 method 1Method 2 method 1 XX--

22 72.34 x 72.34 x --1 1 72.44 72.44

SS22 0.1 s 0.1 s11 0.12 0.12 nn22 5 n 5 n11 4 4 AnswerAnswer t tabulated at degree of freedom nt tabulated at degree of freedom n11 +n +n2 2 – 2 = 7 – 2 = 7

2.3652.365 Sp = 0.109Sp = 0.109 t calculated = 1.36t calculated = 1.36 The null hypothesis is correct and the difference is not The null hypothesis is correct and the difference is not

significantsignificant

Validation Of the analytical Validation Of the analytical procedureprocedure

Validation of an analytical method is the process by which it Validation of an analytical method is the process by which it is established, by laboratory studies that the performance is established, by laboratory studies that the performance characteristic of the method meet the requirements for the characteristic of the method meet the requirements for the intended analytical applications. intended analytical applications.

Validation implies one is able to document that a process is Validation implies one is able to document that a process is correct or is suited for its intended use.correct or is suited for its intended use.

The difference between validation and verificationThe difference between validation and verification is that validation is ensuring "you built the right product" is that validation is ensuring "you built the right product"

and verification is ensuring "you built the product rightand verification is ensuring "you built the product right Verification is usually an internal quality process of Verification is usually an internal quality process of

determining compliance with a regulation, standard, or determining compliance with a regulation, standard, or specificationspecification

The parameters that should be considered during The parameters that should be considered during the validation of analyticalthe validation of analytical procedures:procedures:

1. Specificity1. Specificity is the ability to measure accurately and is the ability to measure accurately and

specifically the analyte in the presence of components that may be specifically the analyte in the presence of components that may be expected to be present in the sample matrix.expected to be present in the sample matrix.

An investigation of specificity should be conducted during the An investigation of specificity should be conducted during the validation of identification tests, the determination of impurities and validation of identification tests, the determination of impurities and the assay. The procedures used to demonstrate specificity will the assay. The procedures used to demonstrate specificity will depend on the intended objective of the analytical procedure depend on the intended objective of the analytical procedure

1.1. Identification1.1. Identification

Suitable identification tests should be able to discriminate Suitable identification tests should be able to discriminate between compounds of closely related structures which are likely to between compounds of closely related structures which are likely to be present. The discrimination of a procedure may be confirmed by be present. The discrimination of a procedure may be confirmed by obtaining positive results (perhaps by comparison with a known obtaining positive results (perhaps by comparison with a known reference material) from samples containing the analyte, coupled reference material) from samples containing the analyte, coupled with negative results from samples which do not contain the with negative results from samples which do not contain the analyte. In addition, the identification test may be applied to analyte. In addition, the identification test may be applied to materials structurally similar to or closely related to the analyte to materials structurally similar to or closely related to the analyte to confirm that a positive response is not obtained. confirm that a positive response is not obtained.

1.2. Assay and Impurity Test(s)1.2. Assay and Impurity Test(s). . Critical separations in chromatography should be investigated at Critical separations in chromatography should be investigated at

an appropriate level. For critical separations, specificity can be an appropriate level. For critical separations, specificity can be demonstrated by the resolution of the two components which elute demonstrated by the resolution of the two components which elute closest to each other. closest to each other.

1.2.1 Impurities are available1.2.1 Impurities are available

For the assay, this should involve demonstration of the For the assay, this should involve demonstration of the discrimination of the analyte in the presence of impurities discrimination of the analyte in the presence of impurities and/or excipients; practically, this can be done by spiking and/or excipients; practically, this can be done by spiking pure substances with appropriate levels of impurities and/or pure substances with appropriate levels of impurities and/or excipients and demonstrating that the assay result is excipients and demonstrating that the assay result is unaffected by the presence of these materials (by comparison unaffected by the presence of these materials (by comparison with the assay result obtained on unspiked samples). with the assay result obtained on unspiked samples).

For the impurity test, the discrimination may be established For the impurity test, the discrimination may be established by spiking drug substance or drug product with appropriate by spiking drug substance or drug product with appropriate levels of impurities and demonstrating the separation of these levels of impurities and demonstrating the separation of these impurities individually and/or from other components in the impurities individually and/or from other components in the sample matrix. sample matrix.

1.2.2 Impurities are not available1.2.2 Impurities are not available specificity may be demonstrated by comparing the test specificity may be demonstrated by comparing the test

results of samples containing impurities or degradation results of samples containing impurities or degradation products to a second well-characterized procedure e.g.: products to a second well-characterized procedure e.g.: pharmacopoeias method or other validated analytical pharmacopoeias method or other validated analytical procedure. Peak purity tests may be useful to show that the procedure. Peak purity tests may be useful to show that the analyte chromatographic peak is not Attributable to more analyte chromatographic peak is not Attributable to more than one component (e.g., diode array, mass spectrometry).than one component (e.g., diode array, mass spectrometry).

2. Linearity2. Linearity

It may be demonstrated directly on the drug substance (by It may be demonstrated directly on the drug substance (by dilution of a standard stock solution) and/or separate weightings' dilution of a standard stock solution) and/or separate weightings' of synthetic mixtures of the drug product components, using the of synthetic mixtures of the drug product components, using the proposed procedure. proposed procedure.

Linearity should be evaluated by visual inspection of a plot of Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. If there signals as a function of analyte concentration or content. If there is a linear relationship, test results should be evaluated by is a linear relationship, test results should be evaluated by appropriate statistical methods, for example, by calculation of a appropriate statistical methods, for example, by calculation of a regression line by the method of least squares. In some cases, to regression line by the method of least squares. In some cases, to obtain linearity between assays and sample concentrations, the obtain linearity between assays and sample concentrations, the test data may need to be subjected to a mathematical test data may need to be subjected to a mathematical transformation prior to the regression analysis. transformation prior to the regression analysis.

Y = mX + b where Y is test result, m slope, X concentration, b Y = mX + b where Y is test result, m slope, X concentration, b interceptintercept

m= m= ΣΣ xy - xy - ΣΣx x ΣΣy/ny/n ΣΣ x x22 - ( - (ΣΣ x) x)22 /n /n b= yb= y-- - mx - mx- - σσxx Correlation coefficient (r) = m ---- Correlation coefficient (r) = m ---- σσyy r equal 1 or nearly 1r equal 1 or nearly 1

For the establishment of linearity, a minimum of 5 concentrations For the establishment of linearity, a minimum of 5 concentrations is recommended.is recommended.

Chromatogram of different concentration of norfloxacin from 0.1 – 0.8 mg% and the same concentration of salicylic acid 2 mg % at 274 nm

A calibration curve plot showing limit of detection (LOD), limit of quantification (LOQ), dynamic range, and limit of linearity (LOL).

3. Range3. Range

The range of an analytical method is the interval The range of an analytical method is the interval between the upper and lower levels of the analyte which between the upper and lower levels of the analyte which could be determined with an acceptable degree of linearity, could be determined with an acceptable degree of linearity, accuracy and precision.accuracy and precision.

The following minimum specified ranges should be The following minimum specified ranges should be considered: for the assay of a drug substance or a finished considered: for the assay of a drug substance or a finished (drug) product: normally from 80 to 120 percent of the test (drug) product: normally from 80 to 120 percent of the test concentration; for content uniformity, covering a minimum of concentration; for content uniformity, covering a minimum of 70 to 130 percent of the test concentration, unless a wider 70 to 130 percent of the test concentration, unless a wider more appropriate range, based on the nature of the dosage more appropriate range, based on the nature of the dosage form (e.g., metered dose inhalers), is justified; for dissolution form (e.g., metered dose inhalers), is justified; for dissolution testing: +/-20 % over the specified range; e.g., if the testing: +/-20 % over the specified range; e.g., if the specifications for a controlled released product cover a region specifications for a controlled released product cover a region from 20%, after 1 hour, up to 90%, after 24 hours, the from 20%, after 1 hour, up to 90%, after 24 hours, the validated range would be 0-110% of the label claim. validated range would be 0-110% of the label claim.

For the determination of an impurity: from the reporting level For the determination of an impurity: from the reporting level of an impurity 1 to 120% of the specification of an impurity 1 to 120% of the specification

4. ACCURACY4. ACCURACY Accuracy is the degree of agreement between the experimental result and the true Accuracy is the degree of agreement between the experimental result and the true

value. or most probable value. It expresses the correctness of the result. value. or most probable value. It expresses the correctness of the result. Accuracy should be established across the specified range of the analytical procedure. Accuracy should be established across the specified range of the analytical procedure.

Measures to express accuracyMeasures to express accuracy

A. Absolute error (d)A. Absolute error (d) The difference between the analytical result and the true value The difference between the analytical result and the true value d= x-µ where x observed value, µ true valued= x-µ where x observed value, µ true value Absolute error has no significance when separated from true or observed value .so we Absolute error has no significance when separated from true or observed value .so we

use relative errors.use relative errors.

ExampleExample Atomic absorption analysis of AsAtomic absorption analysis of As+3+3 and pb and pb+2+2 in a sample yield the following results in a sample yield the following results AsAs+3+3 = 600 µg.ml-1 d = 5 µg. ml-1 = 600 µg.ml-1 d = 5 µg. ml-1 pbpb+2+2 = 9 µg.ml-1 d = 0.3 µg.ml-1 = 9 µg.ml-1 d = 0.3 µg.ml-1 From absolute error AsFrom absolute error As+3+3 results is less accurate than pb results is less accurate than pb+2+2 results but is not true as we results but is not true as we

see from relative error.see from relative error.

B.Relative error (E rel)B.Relative error (E rel) E rel = d/µ .100E rel = d/µ .100 E rel E rel AsAs+3+3 = 5/600 x100 =0.83% E rel pb = 5/600 x100 =0.83% E rel pb+2+2 = 0.3/9 x100 = 3.3 % = 0.3/9 x100 = 3.3 % From the aforementioned, it is obvious that AsFrom the aforementioned, it is obvious that As+3+3 results is more accurate, although it results is more accurate, although it

has larger absolute error.has larger absolute error. C. Recovery % (Relative accuracy)C. Recovery % (Relative accuracy) = x/µ .100 = amount found/ amount claimed= x/µ .100 = amount found/ amount claimed or calculated .100 or calculated .100

Several methods of determining accuracy are available: Several methods of determining accuracy are available:

For For Drug SubstanceDrug Substance a) Application of an analytical procedure to an analyte of known purity (e.g. a) Application of an analytical procedure to an analyte of known purity (e.g.

reference material); reference material); Comparison of the results of the proposed analytical procedure with those Comparison of the results of the proposed analytical procedure with those

of a second well-characterized procedure, the accuracy of which is stated of a second well-characterized procedure, the accuracy of which is stated and/or defined. and/or defined.

For Drug ProductFor Drug Product a) Application of the analytical procedure to synthetic mixtures of the drug a) Application of the analytical procedure to synthetic mixtures of the drug

product components to which known quantities of the drug substance to be product components to which known quantities of the drug substance to be analyzed have been added; analyzed have been added;

b) In cases where it is impossible to obtain samples of all drug product b) In cases where it is impossible to obtain samples of all drug product components, it may be acceptable either to add known quantities of the components, it may be acceptable either to add known quantities of the analyte to the drug product or to compare the results obtained from a analyte to the drug product or to compare the results obtained from a second, well characterized procedure, the accuracy of which is stated second, well characterized procedure, the accuracy of which is stated and/or defined and/or defined

For Impurities (Quantization) For Impurities (Quantization) Accuracy should be assessed on samples (drug substance/drug product) Accuracy should be assessed on samples (drug substance/drug product)

spiked with known amounts of impurities. spiked with known amounts of impurities. In cases where it is impossible to obtain samples of certain impurities In cases where it is impossible to obtain samples of certain impurities

and/or degradation products, it is considered acceptable to compare results and/or degradation products, it is considered acceptable to compare results obtained by an independent procedure. It should be clear how the obtained by an independent procedure. It should be clear how the individual or total impurities are to be determined e.g., weight/weight or individual or total impurities are to be determined e.g., weight/weight or area percent, in all cases with respect to the major analyte. area percent, in all cases with respect to the major analyte.

Accuracy should be assessed using a minimum of 9 determinations over a Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range (e.g. 3 minimum of 3 concentration levels covering the specified range (e.g. 3 concentrations /3 replicates each of the total analytical procedure). concentrations /3 replicates each of the total analytical procedure).

Accuracy should be reported as percent recovery by the assay of known Accuracy should be reported as percent recovery by the assay of known added amount of analyte in the sample or as the difference between the added amount of analyte in the sample or as the difference between the mean and the accepted true value together with the confidencemean and the accepted true value together with the confidence intervals. intervals.

Low AccuracyHigh Precision

High AccuracyLow Precision

High AccuracyHigh Precision

Examples of Precision and Accuracy:

5. PRECISION5. PRECISION Precision is the degree of agreement among a series of Precision is the degree of agreement among a series of

measurements of the same quantity; it is a measure of the measurements of the same quantity; it is a measure of the reproducibility of results rather than their correctnessreproducibility of results rather than their correctness

Measures to express precisionMeasures to express precision

standard deviation (sstandard deviation (s) )

Is calculated by using equation 1, where Σ represents Is calculated by using equation 1, where Σ represents summation, xi represents each of the individual analytical summation, xi represents each of the individual analytical results, a; is the average of the results, and N is the number results, a; is the average of the results, and N is the number of replicate assays.of replicate assays.

The standard deviation is a popular estimate of the error in The standard deviation is a popular estimate of the error in an analysis because it has statistical significance whenever an analysis because it has statistical significance whenever the results are normally distributed. Most analytical results the results are normally distributed. Most analytical results exhibit normal (Gaussian) behavior, following the exhibit normal (Gaussian) behavior, following the characteristic bell-shaped curve. If the results are normally characteristic bell-shaped curve. If the results are normally distributed, 68.3 percent of the results can be expected to fall distributed, 68.3 percent of the results can be expected to fall within the range of plus or minus one standard deviation of within the range of plus or minus one standard deviation of the mean as a result of random error. the mean as a result of random error.

Chromatogram of the same concentration ofNorfloxacin; (a) 5 mg% and the same concentration of

Salicylic acid, (d) 2 mg% id at 274 nm.

b. Relative standard deviationb. Relative standard deviation S rel = s /x- .100 also called coefficient of variationS rel = s /x- .100 also called coefficient of variation

C .Confidence limitsC .Confidence limits at a given probability level are values greater than and less than the average, between which at a given probability level are values greater than and less than the average, between which

the results are statistically expected to fall a given percentage of the time.the results are statistically expected to fall a given percentage of the time. µ=x- ± ts\ √nµ=x- ± ts\ √n We might use this to estimate the probability that the population mean (µ) lies within a certain We might use this to estimate the probability that the population mean (µ) lies within a certain

region centered at x- region centered at x- ExampleExample A pharmacist determined the % of vitamin C in Rino C Tablets, obtaining the following results, A pharmacist determined the % of vitamin C in Rino C Tablets, obtaining the following results, X- = 15.3 s= 0.1 n= 4X- = 15.3 s= 0.1 n= 4 Calculate the confidence limit of the mean at probability90% and 99% Calculate the confidence limit of the mean at probability90% and 99% (t =2.353 at 90 % probability and, 5.841 at probability 99%)(t =2.353 at 90 % probability and, 5.841 at probability 99%) µ = 15.3 ± 2.353 x 0.1/√4 µ = 15.3 ± 0.12 µ = 15.3 ± 2.353 x 0.1/√4 µ = 15.3 ± 0.12 µ = 15.3 ± 5.841 x 0.1/√4 µ = 15.3 ± 0.29µ = 15.3 ± 5.841 x 0.1/√4 µ = 15.3 ± 0.29

Repeatability should be assessed usingRepeatability should be assessed using: : a) A minimum of 9 determinations covering the specified range for the procedure (e.g. 3 a) A minimum of 9 determinations covering the specified range for the procedure (e.g. 3

concentrations/3 replicates each) concentrations/3 replicates each) Or b) A minimum of 6 determinations at 100% of the test concentration. Or b) A minimum of 6 determinations at 100% of the test concentration.

Recommended DataRecommended Data The standard deviation, relative standard deviation (coefficient of variation) and The standard deviation, relative standard deviation (coefficient of variation) and confidence interval should be reported for each type of precision investigated. confidence interval should be reported for each type of precision investigated.

6. Detection Limit6. Detection Limit Detection limit is the smallest concentration or amount of substances which can be Detection limit is the smallest concentration or amount of substances which can be

reported with a specified degree of certainty by a definite, complete analytical procedure. reported with a specified degree of certainty by a definite, complete analytical procedure. Several approaches for determining the detection limit are possible, depending on whether Several approaches for determining the detection limit are possible, depending on whether

the procedure is a non-instrumental or instrumental. the procedure is a non-instrumental or instrumental.

6.1. Based on Visual Evaluation6.1. Based on Visual Evaluation The detection limit is determined by the analysis of samples with known The detection limit is determined by the analysis of samples with known

concentrations of analyte and by establishing the minimum level at which the analyte can concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected. be reliably detected.

6.2. Based on Signal-to-Noise6.2. Based on Signal-to-Noise This approach can only be applied to analytical procedures which exhibit baseline This approach can only be applied to analytical procedures which exhibit baseline

noise. noise. Determination of the signal-to-noise ratio is performed by comparing measured signals Determination of the signal-to-noise ratio is performed by comparing measured signals

from samples with known low concentrations of analyte with those of blank samples and from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A establishing the minimum concentration at which the analyte can be reliably detected. A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit. detection limit.

6.3 Based on the Standard Deviation of the Response and the Slope6.3 Based on the Standard Deviation of the Response and the Slope

The detection limit (DL) may be expressed as: The detection limit (DL) may be expressed as: DL = 3.3s/ S DL = 3.3s/ S Where;Where; s= the standard deviation of the response s= the standard deviation of the response S = the slope of the calibration curve S = the slope of the calibration curve The slope S may be estimated from the calibration curve of the analyte. The slope S may be estimated from the calibration curve of the analyte.

The estimate of s may be carried out in a variety of ways, for exampleThe estimate of s may be carried out in a variety of ways, for example:: 6.3.1 Based on the Standard Deviation of the Blank6.3.1 Based on the Standard Deviation of the Blank

Measurement of the magnitude of analytical background response is Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and performed by analyzing an appropriate number of blank samples and calculating the standard deviation of these responses.calculating the standard deviation of these responses.

6.3.2 Based on the Calibration Curve6.3.2 Based on the Calibration Curve

A specific calibration curve should be studied using samples containing an A specific calibration curve should be studied using samples containing an analyte in the range of DL. The residual standard deviation of a regression line analyte in the range of DL. The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the or the standard deviation of y-intercepts of regression lines may be used as the standard deviation. standard deviation.

6.4 Recommended Data6.4 Recommended Data

The detection limit and the method used for determining the detection limit The detection limit and the method used for determining the detection limit should be presented. If DL is determined based on visual evaluation or based on should be presented. If DL is determined based on visual evaluation or based on signal to noise ratio, the presentation of the relevant chromatograms is signal to noise ratio, the presentation of the relevant chromatograms is considered acceptable for justification. considered acceptable for justification.

7. Quantization Limit7. Quantization Limit

the minimum level at which the analyte can be quantified with the minimum level at which the analyte can be quantified with acceptable accuracy and precision. acceptable accuracy and precision.

Based on Signal-to-Noise ApproachBased on Signal-to-Noise Approach

Determination of the signal-to-noise ratio is performed by Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low comparing measured signals from samples with known low concentrations of analyte with those of blank samples and by concentrations of analyte with those of blank samples and by establishing the minimum concentration at which the analyte can be establishing the minimum concentration at which the analyte can be reliably quantified. A typical signal-to-noise ratio is 10:1 reliably quantified. A typical signal-to-noise ratio is 10:1

Based on the Standard Deviation of the Response and the Based on the Standard Deviation of the Response and the SlopeSlope

The quantization limit (QL) may be expressed as: The quantization limit (QL) may be expressed as: QL = 10s/ S QL = 10s/ S Where Where s= the standard deviation of the response s= the standard deviation of the response S = the slope of the calibration curve S = the slope of the calibration curve The slope S may be estimated from the calibration curve of the The slope S may be estimated from the calibration curve of the

analyte. analyte.

The estimate of s may be carried out in a variety of ways The estimate of s may be carried out in a variety of ways for example:for example:

Based on Standard Deviation of the BlankBased on Standard Deviation of the Blank Measurement of the magnitude of analytical Measurement of the magnitude of analytical

background response is performed by analyzing an background response is performed by analyzing an appropriate number of blank samples and calculating the appropriate number of blank samples and calculating the standard deviation of these responses.standard deviation of these responses.

Based on the Calibration CurveBased on the Calibration Curve A specific calibration curve should be studied using A specific calibration curve should be studied using

samples, containing an analyte in the range of QL. The samples, containing an analyte in the range of QL. The residual standard deviation of a regression line or the residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may standard deviation of y-intercepts of regression lines may be used as the standard deviation.be used as the standard deviation.

Recommended DataRecommended Data The quantization limit and the method used for The quantization limit and the method used for

determining the quantization limit should be presented. determining the quantization limit should be presented. The limit should be subsequently validated by the analysis The limit should be subsequently validated by the analysis of a suitable number of samples known to be near or of a suitable number of samples known to be near or prepared at the quantization limit. prepared at the quantization limit.

8. Robustness8. Robustness

Robustness of an analytical procedure is a measure of Robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate its capacity to remain unaffected by small but deliberate variations in method parameters. It should show the variations in method parameters. It should show the reliability during its usage.reliability during its usage.

One consequence of the evaluation of robustness should One consequence of the evaluation of robustness should be that a series of system suitability parameters (e.g., be that a series of system suitability parameters (e.g., resolution test) is established to ensure that the validity of resolution test) is established to ensure that the validity of the analytical procedure is maintained whenever used.the analytical procedure is maintained whenever used.

Examples of typical variations are: stability of analytical Examples of typical variations are: stability of analytical

solutions, extraction time. In the case of liquid solutions, extraction time. In the case of liquid chromatography, examples of typical variations are influence chromatography, examples of typical variations are influence of variations of pH in a mobile phase, influence of variations of variations of pH in a mobile phase, influence of variations in mobile phase composition, different columns (different lots in mobile phase composition, different columns (different lots and/or suppliers), temperature, flow rate.and/or suppliers), temperature, flow rate.

In the case of gas-chromatography, examples of typical In the case of gas-chromatography, examples of typical

variations are different columns (different lots and/or variations are different columns (different lots and/or suppliers), temperature, flow rate. suppliers), temperature, flow rate.

9. Ruggedness9. Ruggedness Ruggedness of an analytical method is the Ruggedness of an analytical method is the

degree of reproducibility of the results obtained by degree of reproducibility of the results obtained by the analysis of the same sample under a variety of the analysis of the same sample under a variety of normal test conditions for example different normal test conditions for example different laboratory, different analysts, different instruments, laboratory, different analysts, different instruments, and different reagentsand different reagents..

10. System suitability testing10. System suitability testing The tests are based on the concept that the The tests are based on the concept that the

equipment, electronics, analytical operations and equipment, electronics, analytical operations and samples to be analyzed constitutes an integral samples to be analyzed constitutes an integral system that can be evaluated as such.system that can be evaluated as such.

SensitivitySensitivity Sensitivity is the change in measured value Sensitivity is the change in measured value

resulting from a concentration change of one unit.resulting from a concentration change of one unit.