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Analytical Aspect of Quality Control and Quality assurance

For pharmacy students By Dr. Abdalla Ahmed El shanawany Professor of Medicinal Chemistry Vice Dean of Faculty of Pharmacy Zagazig University 2010

• Chapter 1: Drug registration and assessment 1.1. Summary of particulars 1.2. Chemical and pharmaceutical documentation 1.3. Reports of Experimental and biological studies 1.4. Report on Clinical Studies 1.5. Company profile • Chapter 2: The Analytical Problem 2.1. Sampling/Sample Handling 2.2. Experimental errors 2.3. Choice of methods of an analysis 2.4. Statistics of data analysis 2.5. Validation of analytical procedure

Chapter 3: Drug stability and degradation product 3.1. Chemical purity and its control 3.2. International pharmacopoeia and official monograph 3.3. Radiopharmaceutical 3.4. Stability indicating assay Chapter 4: Function group analysis 4.1. Classical analysis 4.2. Instrumental analysis 4.2.1. Spectral methods 4.2.2. Electro analytical methods Chapter 5: Automation in pharmaceutical analysis 5.1. Flow injection analysis 5.2. HPLC 5.3. Gas chromatography. 5.4. Mass spectroscopy

Chapter 6: Determination of active ingredients in different dosage forms and in biological fluids 6.1. Determination of active ingredients in tablets 6.2. Determination of active ingredients in capsules 6.3. Determination of active ingredients in semisolid 6.4. Determination of active ingredients in eye drops 6 5. Determination of active ingredients in injection 6.6. Determination of active ingredients in Suppositories. 6.7. Determination of active ingredients in aerosols inhalation 6.8. Radioimmunoassay


Chapter 7: Quality assurance of pharmaceuticals 7.1. Good Manufacturing Practice G.M.P 7.2. National and international organization 7.3. Departments in pharmaceutical companies 7.4. ISO and BSI

Intended Learning Outcomes of Course (lLOs)

A -Knowledge and Understanding • Chemical QC/QA is only a part and aspect of total QC/QA that should go hand in hand with physical and biological aspects and all aspects should work in perfect synchronization within process control, validation and other aspects of G.M.Ps. • That organization of data and documentation coupled with a working knowledge of relevant statistical methods is mandatory

B- Intellectual Skills • Should be able to choose and develop suitable analytical methodology to suit the purpose at hand so as to face various QC/QA settings e.g.; raw material/ single component/partially degraded dosage forms/ multicomponent dosage forms . • -Given the chemical structure of the ingredients selection of analytical procedures should be based on a hybrid methodology basis.

To be able to apply previously known analytical methods to the area of drug analysis D. The capability to analyze and find an effective solution for a given .Professional and Practical Skills .General and Transferable Skills Work effectively as part of a team to collect data and/or to produce reports and presentations.C.

• All licenses and certificates are issued by the appropriate licensing authority which is department of health and social security.Drug registration and assessment • Any pharmaceutical product before it is made available to the market needs evaluation and registration. • Product licenses are required for the manufacture of all medicine. • A guideline to provide manufacturers with information concerning documentation to be submitted for approval and registration of pharmaceutical products for human use is . medicines division. Also animal test and clinical trial certificate are required. for sale and supply for import and export.

Application form for drug registration • 1. and suggested trade name physical properties of active constituent recommended clinical use. place of manufacture. sale. the license. dose. dosage form. Summary of particulars General information relating applicant. route of administration. and the product • description and name of the product. OTC or by prescription and supply .

1. physicochemical properties. Chemical Data on active ingredient • Nonproprietary or generic name. chemical name. .• 2. key intermediates. structure. Chemical and pharmaceutical documentation • 2. molecular formula. key raw materials. degradation profile. synthesis. analytical specifications and test methods. stability studies. including analytic procedures used in the detection and determination of byproducts.

including quality specifications (requirements) and control methods. which comes in to such contact with the drug Package labeling includes package leaflet. and outer wrapper or carton. • Data on Packaging Materials (Container and Closures) • Detailed information is required about the packaging material. Active ingredient(s) present in the form of salts or hydrates shall be described quantitatively by their total mass and by the mass of the active moiety or moieties of the molecule.• 2. Formulation Report (dosage forms) • Data on Composition • Complete qualitative and quantitative composition of the finished product.2. A leaflet must bear adequate information for use. label on the immediate container. • The package leaflet should consist of factual and scientific information consistent with the application. .

3. Stability Report • The stability report should consist of stability data sheet and a summary.• 2.4. Analytical Report • The manufacturer should submit: • Quality specifications (requirements) and analytical procedures for the dosage form • 2. The tests for stability at each sampling period should be related to the formulation and to the storage condition and the study should include .

number of animals. . concise description of the methodology. Pharmacodynamics • Studies providing the primary basis for clinical trials of the drug. mechanism of action. • 3. minimum effective dose emphasizing adequate description of dose-effect relationships that produce pharmacological responses in each species of animal investigated. information route of administration. results.1. Animal Pharmacology • The manufacturer should furnish a summary of the observations and conclusions Showing the animal species. conclusions and an overall evaluation of the pharmacodynamic and pharmacokinetics properties of the drug based on the findings in laboratory animals or in invitro systems.• 3.2. doses. Reports of Experimental and biological studies. (Pre clinical studies) • • 3.

number. age. Cumulative MIC tables are highly desirable. significant observations and conclusions. metabolism.• 3. and excretion. treatment regimen duration of treatment. detailed description and analysis based on the available completed microbiologic studies should be provided. including methods used together with a discussion and evaluation of the results. . sex. weight and strain of animals. enzyme inhibition. distribution. Microbiology (for anti-microbial agents only) • Summaries of all microbiologic studies. Under each of the following headings. information on dosage formulation. • 3.5. parameters evaluated. Pharmacokinetics • Pharmacokinetics Studies concerning absorption. enzyme induction.3. • 3. Toxicological Data • Summary of toxicological studies preferably should be presented in tables which indicate species. route(s) of administration.4.

Clinical Pharmacology: • Pharmacodynamics • Intended drug effect. Report on Clinical Studies • Drug clinical trials shall be conducted in institutions legally certified for drug clinical trials. The investigator responsible for a drug clinical trial shall. healthy and sick. If a clinical trial has to be conducted by an institution not yet certified. age groups. mechanism of action. a special approval by the State Food and Drug Administration should be obtained. number of volunteers. And the effect of drugs on various organic functions.• 4. methodology. drug . in accordance with the relevant provisions. timely report adverse events occurred in the process of the clinical trial to the State Food and Drug Administration. studies on the relationship of between dose of drug and response in patient. optimal dose Studies of single and multiple dose.1. • 4.

elimination of the drug and also report on metabolic studies.• Pharmacokinetics • Studies on the kinetics. Bioavailability Report • Bioequivalence report is required for those oral dosage forms of drugs which are known to pose bioavailability problem. distribution plasma concentration. Physicochemical properties which may act on absorption and distribution should be stated. should also give information on the pharmacological properties of the particular combination that is being considered. biotransformation. half. in addition to details on their individual components. • 4. • Reports on combined preparations. where systemic absorption is a requirement for their efficacy. .2. The methods of assay or determination should be specified. protein binding.

and also give an overall discussion and evaluation of the safety. • • • . etc. Clinical Trials The summary should concisely set out the clinical properties of the drug. It should provide information on patient population number of patients. efficacy. dosage formulation. dosages. and assessment and comparison of the benefit/risk ratio of the drug in relation to related drugs or others used as standards in controlled clinical trials. etc. methods. Special emphasis should be put on that documentation which lends support to the cited indications. and conclusion providing a discussion of the benefits and risks of the drug under the conditions of use recommended.3. doses. The conclusion should at least consider the following points: comparison of the expected clinical benefits with possible adverse effects. adverse reactions and contraindications of the drug based on the findings of available completed clinical trials.• • 4.

Back ground information The manufacturer should submit background information about the company indicating.C. .. Raw & packaging materials Q.C.1. Standard operation manual. Year of establishment. Master file and batch production record system Product specifications. The types of Q. The major Q.• • • • 5. Qualification and experience of Q. 5. Pyrogen test.C. GMP procedure. D. physicochemical tests. E. B.3. List of pharmaceuticals produced by the manufacturer (specify those which are the manufacturer's innovation). 5.C. 5. Whether it has Good laboratory Practice (GLP) Procedure. Biological assay. Development since establishment.C. acute toxicity test. Total working force.2. Ownership. Sterility test . tests performed. Whether it performs.C. Production Unit The manufacturer should submit information on the production unit and should also indicate whether the company has the following. and Finished product Q. Instruments available. Microbiological assay etc. personnel • • • • • • • • • • • • • • . C. Quality Control Unit The manufacturer should state: A. In-process Q. Company profile Documents to be supplied by the manufacturer.

Qualification of the personnel engaged in R and D activities Major research areas and achievements attained.6. 5. Supply system The manufacturer should give information on its supply system indicating whether it has at least the following: Cold storage facilities. packaging materials. Separate stores for raw materials.4. Affiliation with other institutes (if there is any) 5. Product Registration and marketing Experience of the manufacturer The manufacturer should submit full information on its marketing experience and Registration status of its products indicating: List of countries to which it exports most of its products.. finished products. Quarantine for raw materials. procedure for supplies control.• • • 5. etc.. labels etc. Research and Development Unit (R and D) The manufacturer should give detailed information on at least the following major points: The year R and D was initiated. Separate room for weighing raw materials. List of countries in which its products are registered • • • • • • • • • • .5.

3 Choice of methods of an analysis 2.5 Validation Of analytical procedure.1 Sampling/Sample Handling 2. . Preparation of the sample for analysis 3.• Chapter 2: Analytical problem 2. • There are three basic activities involved in solving an analytical problem: 1. Collection of the applicable sample 2.2 Experimental errors 2.4 Statistics of data analysis 2. Analysis using appropriate methods.

and it is the responsibility of the study director to ensure sample homogeneity. Many of these situations can be avoided if samples are collected according to a rational plan that gives some assurance that the sample delivered to the laboratory represents the composition of the parent lot.S. The important point in this example is to show that sampling error can play a very significant part in the overall error in the analytical system. a serious problem may arise Involving questions of competence and credibility. One of the most common causes of differing analytical results can be traced back to non representative ness of different samples. It is easy to anticipate that this could occur during the conduct of a multi-lab study. Environmental Protection Agency conducted studies during remediation projects to determine the sources of variation in the sampling and analytical procedures. For example when the U.• • • • • • 2. and found that the amount of variation from sampling was approximately 80% of the total variation while the amount of variation from the analytical procedures was 20%. When a significant difference in results occurs between laboratories that have analyzed supposedly the same sample. .1 Sampling/Sample Handling Sample is a representative portion selected from the bulk.

Distortions introduced at this point will carry through the analysis and adversely affect the final results and the conclusions drawn from them. • The best analysis can give misleading information if the test portion analyzed does not represent the sample or the lot from which it was taken. • Heterogeneous materials.and exhibit a range of particle size. readily yields a representative sample. such as powdered materials and granules always exhibit segregation of the particles during handling . such as a liquid solution. and the flow properties of the powder depend upon the particle size distribution .• Analytical sample • Analytical sample is a small portion selected from the sample. • A homogenous material.

and then the cone is flattened and divided into four equal parts by forming two perpendicular diameters. that material in the other two quarters being set aside . Coning and quartering The powder is deposited on a flat surface by shovel. coning and qartering . and then separated into two equal piles using a shovel and throwing alternative shovels to opposite sides.• • Two techniques for obtaining good sample from powdered material: Long pile method The gross sample is arranged in long pile. Long pile 2.the coning and quartering process is continued until a sample of the requisite size is obtained. The material from two diagonally opposed quarters is combined and formed into another cone. One half is discarded and the other again separated in the same manner until the required sample size is obtained.

The selection of n is of course critical. but it is apt to be laborious and time consuming.all numbered have an equal number of digits. bottles. there are two general sampling approaches • Random sampling The units are numbered serially . • Systematic sampling A more widely used time saving technique in which every nth unit is selected to constitute the sample.. Then numbers are selected in some random manner from the random table. It is an effective way to obtain unbiased. and the corresponding units are taken for the sample. It is biased to some . package of ampoules.• When the populations consist of discrete units. representative samples. as the interval between selected units must not correspond to any periodicity in the population. such as drums of solvent. etc.

where r is the sample size. • • • • • . Preparation of a composite laboratory sample (if multiple units are submitted for analysis) 2.5√ N. 1. N is the quantity to be sampled The p plan For homogenous and the sample for identity P= 0. or Representative portions of units are mixed to form a uniform mixture.• • • • • • 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. Compositing saves analytical time and in some types of contract testing it May be the procedure specified. A composite laboratory sample is one in which the individual units. N is the quantity to be sampled There are generally two choices specify the manner in which an analytical sample should be taken. Examination of individual units.4√ N The r plan For heterogonous and vegetable drug as raw materials r= 1. Portions are then taken from the composite for analysis Compositing can Best be used when homogeneity is not a significant problem concern.

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

Dry particulate materials can be reduced in volume by use of a splitter. stirred. In particular.• • Sample Preparation for Analysis Every type of material that is to be prepared for analysis presents its own practical difficulties. grinders and cutters. Screening can be used to improve the efficiency of particle size reduction. A variety of implements and machines are available for sample disintegration. and of course. and by the distribution of the analyte in the sample. such as mills. • • . from contaminating the laboratory space and any subsequent samples that are ground. Single phase liquids can generally be mixed. shaken. Care in their use is necessary to prevent loss of dust or change in composition through the partial separation of components. Even seemingly homogeneous materials such as liquids may be subject to Sedimentation or stratification. care must be taken to prevent dust or related substances as carry over. The requirements for suitable sample preparation are dictated by the consistency and the chemical characteristics of the analyte and the matrix. followed by mixing to attain homogeneity. the grinding equipment must be meticulously cleaned between samples.

In other words. validation of the method of sample preparation and storage. most certainly. • Loss or gain of moisture during manipulation can be a problem. • Plastic containers can retain contaminants. • When volatile organic constituents are present in samples. Loss can be minimized by keeping samples covered with plastic or aluminum foil. in order to prevent . sample manipulation may not be possible. while the rest of the sample is transferred with apparent ease. or may be severely restricted.• Glass containers and laboratory apparatus can adsorb certain materials and may require surface treatment. validation of a method of analysis includes. such as animal hairs. A cold product can be protected from gaining moisture by allowing the sample to come to room temperature before preparation begins.

differences between the standard values and the results obtained by the new method are then treated as error in the latter. • One can attempt to minimize errors but cannot eliminate them completely • Random errors small fluctuations introduced in nearly all analyses. repeated measurements of the same property often differ even if they are performed on a single instrument that is calibrated and operated properly. Arise from variation of external conditions over which the observer has no control For example. . the % composition of a standard sample certified by the national Bureau of standards may be treated as correct in evaluating a new analytical method.• • • Experimental Errors Random (in determinant) and systematic (determinant) errors The term error as used here refers to the numerical difference between a measured value and the true value. For example.

3. instrumental and methodic 1. pipette. The measurement lacks accuracy. Methodic: Errors are those inherent in the method itself. It is even possible that repeated measurements with this broken instrument will give reproducible results (high precision). personal errors: Are those due to the carelessness of the observer and include such things as misreading a burette or overshooting the end point. • • • • . burettes. instability of the reaction product or impurities in the reagents. and mathematic error in calculations. Systematic errors have been classified as personal.g. An example of a systematic error is improper calibration of an instrument. flasks. but every one of them will deviate from the true value (low accuracy). or loss in weight during washing and filtration. Instrumental error: Error due to the instruments themselves and include the use of uncalibrated equipments e. 2. incomplete reaction or side reaction.• Systematic errors Systematic errors cause the results to vary from the correct value in a predictable manner and can often be identified and corrected. Weights.

fixed amount of the suitable standard is added with different amount of the sample to minimize the error during introduction of the sample. • 2-Use of blank experiments Carrying out a separate determination without the sample under the same experimental condition to overcome the effect of impurities introduced through reagents.Minimization of the errors • 1-Calibration of the equipment and application of correction factor. All of the equipments should be corrected and appropriate correction factor applied. • . Use of internal standard It is used in HPLC and GC. The difference between the analytical results for the sample with and without the added standard gives the recovery of the amount of added constituent. 4. solvents and vessels. • • 3. Standard addition method A known amount of the constituent being determined (standard) is added to the sample which is then analyzed for the total amount of the constituent present.

since the analysis is based on reactions or properties share by several compounds. 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 gravimetric methods and use other sensitive methods as HPLC or spectrophotometry. • • . there are no generally applicable rules that can be applied. • • Factors plays an important role in the selection of the analytical methods of analysis.Different components which are present in the sample The chemical structure of these substances should be known because they may interfere with the method. the choice of method is thus a matter of judgment. 1-Concentration range of the sample to be determined. especially in small and vital substances as hormones. in this case prior separation or selective method should be used. 2-Degree of accuracy and precision required The accuracy and precision required are of vital importance in the choice of an analytical method and its performance. 3. On other hand if the analyte is a major component of the sample the classical method may be preferable. and the ability to make it will come only with experience. Unfortunately.• Choice of methods of an analysis 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 information. Such judgment is difficult.

plays an important role in the selection of the analytical method. 5-Number of the sample to be analyzed If there are many considerable times can be expended in calibrating instruments. . and whether or not the sample is hygroscopic or efflorescent and what sort of treatment is sufficient to decompose or dissolve the sample without loss of the analyte. if a few samples are to be analyzed.4-Physical and chemical properties of the substance The analyst should know the state of the substance at ordinary conditions and whether losses by volatility. a longer and more tedious procedure involving a minimum of these preparatory operations may actually prove to be the wiser choice from the economic standpoint. assembling equipments. the cost of these operations can be spread over the large number of analyses. 6-Availability of the instruments and equipments. preparing reagents. On other hand.

Or synthesized from weighed quantities of pure compounds.• Testing the procedure Once a procedure for an analysis has been selected. If the procedure chosen has been the subject 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 it to a type of sample different from that for which it was designed. a preliminary laboratory test is advisable • What are the means by which a new method or a modification of an existing method can be tested for reliability? • 1-Analysis of standard sample The best technique for evaluating an analytical method involves the analysis of one or more standard samples whose composition with respect to the compound of interest is reliably known. The answer depends upon a number of considerations. the problem usually arises as to whether the method can be employed directly without testing. . For this technique to be of value. it is essential that the standards closely resemble the sample to be analyzed with respect to both the concentration range of the analyte and the overall composition. Standard may be purchased from sources such as national Bureau of standards.

Comparable result from the two methods. • Standard addition to the sample Standard addition method. the proposed procedure is tested against portions of the sample to which known amounts of the analyte have been added. The standard addition method may reveal errors arising from the method of treating the sample or from the presence of the other compounds . The effectiveness of the method can be established by evaluating the extent of recovery of the added quantity.• Analysis by other methods The result of an analytical method can sometimes be evaluated by comparison with some entirely different method. no significant difference between the two methods. in addition to being used to analyze the sample itself. serve as presumptive evidence that both are yielding satisfactory results. It should be based on chemical principles that differ considerably from that one under examination.

so that the standard deviation alone is not a good criterion for rejection. then we could calculate x ± 2sx . Statistically speaking.which works well in cases where 3 < N < • • • • • .177 grams belong to the same normal distribution as the other four measurements? . you obtain the following results (in grams): 8. after making only five measurements of our drug. If we had a very large data set. For example. our data set is very small (N < 10).the Q test . you must never throw out a result from a data set unless you have a statistical reason to do so.149 8. and you may be tempted to throw it out of the set. and he then must decide whether to exclude this result from further consideration. However. We will describe only one .148 8.• • Rejection of Data Sometimes a person performing measurements is faced with one result in a set of replicate s which seems to be out of line with others.156 8. we are asking the question: does the measurement of 8.145 8. and then determine if the measurement in question falls outside the confidence interval. However.177 The last measurement seems a bit off. Statisticians have devised many rejection tests for the detection of non-random errors.

A partial list follows: N Q c (90% confidence) 3 0. The Qc value depends on the confidence level and the number of observations in your set.47 9 0.44 10 0.177 Then.177 grams. we see that Q calc = 0.145 = 0 . Q calc = 8 . If Q calc > Q c.8 . then the observation may be rejected.156 / 8.94 4 0. .177 .64 6 0.64.41 Returning to our example. Hence. Q calc  absolute value of the gap between the suspect value and the value closest to it / range of values To calculate Qcalc in our example.8. we must calculate the socalled Qcalc value for this observation. where N = 5. we are justified in rejecting the observation. 177 .66 We now compare this Q calc with a critical value Q c. However.148 8. and then identify the suspect value and the value that is closest to it: 8.66 is indeed greater than QC = 0.149 8.56 7 0.76 5 0.156 8.51 8 0.145 8. If Q calc < Q c. then we must keep the observation. you must indicate in your report that the Q test was used at a 90 % confidence interval.• • • In order to test the value of 8. we display the data in increasing order of numerical value.

.• Testing for significance Suppose that a sample is analyzed by two different methods. yielding means X-1 .and x. each repeated several times and the mean values obtained are different.2 and standard deviation s1 and s2 . and n2 are the number of the individual results obtained by the two methods. First calculate t x1. s t test gives a yes or no answer to the correctness of the null hypothesis with a certain confidence. Procedure: Suppose a sample has been analyzed by two different methods.. find t tabulated at degree of freedom n1+n2 -2 and at the desired probability if the t value in the table less than the • • • • • • • • • . Is the difference between the two values significant? We use null hypothesis which states that the two means are identical and the student. n1.x-2 t =-----------------Sp√ 1/n1 + 1/n2 Where (n1-1) s12 + (n2 -1) s22 Sp (pooled sd) = √ -----------------------n1 +n2 -2 sec.

1 s1 0.34 x-1 72.109 • t calculated = 1.44 S2 0.365 Sp = 0.36 • The null hypothesis is correct and the difference is not significant .• F test (variance ratio) • To decide whether the difference between s1 and s2 is significant F>1 • F = S12/ S22 The larger s in the numerator • Example Aspirin sample was analyzed by two different methods and the following results were given Method 2 method 1 X-2 72.12 n2 5 n1 4 Answer t tabulated at degree of freedom n1 +n2 – 2 = 7 2.

by laboratory studies that the performance characteristic of the method meet the requirements for the intended analytical applications. Validation implies one is able to document that a process is correct or is suited for its intended use. standard. or specification • The parameters that should be considered during the validation of analytical procedures: • . The difference between validation and verification is that validation is ensuring "you built the right product" and verification is ensuring "you built the product right Verification is usually an internal quality process of determining compliance with a regulation.Validation Of the analytical procedure • Validation of an analytical method is the process by which it is established.

1. the identification test may be applied to materials structurally similar to or closely related to the analyte to confirm that a positive response is not obtained. Critical separations in chromatography should be investigated at an appropriate level. In addition. Assay and Impurity Test(s). specificity can be demonstrated by the resolution of the two components which elute closest to each other. • • • • . coupled with negative results from samples which do not contain the analyte. An investigation of specificity should be conducted during the validation of identification tests. The discrimination of a procedure may be confirmed by obtaining positive results (perhaps by comparison with a known reference material) from samples containing the analyte.• 1.2. Specificity • is the ability to measure accurately and specifically the analyte in the presence of components that may be expected to be present in the sample matrix.1. The procedures used to demonstrate specificity will depend on the intended objective of the analytical procedure 1. For critical separations. the determination of impurities and the assay. Identification Suitable identification tests should be able to discriminate between compounds of closely related structures which are likely to be present.

the discrimination may be established by spiking drug substance or drug product with appropriate levels of impurities and demonstrating the separation of these impurities individually and/or from other components in the sample matrix.. • • .2. practically.• • 1.2 Impurities are not available specificity may be demonstrated by comparing the test results of samples containing impurities or degradation products to a second well-characterized procedure e. Peak purity tests may be useful to show that the analyte chromatographic peak is not Attributable to more than one component (e. this should involve demonstration of the discrimination of the analyte in the presence of impurities and/or excipients. mass spectrometry).g.1 Impurities are available For the assay. diode array.: pharmacopoeias method or other validated analytical procedure.2. this can be done by spiking pure substances with appropriate levels of impurities and/or excipients and demonstrating that the assay result is unaffected by the presence of these materials (by comparison with the assay result obtained on unspiked samples). 1. For the impurity test.g.

m slope.Σx Σy/n Σ x2 . X concentration.. for example.mxσx Correlation coefficient (r) = m ---σy r equal 1 or nearly 1 • • • • • • . In some cases. by calculation of a regression line by the method of least squares.• • 2. to obtain linearity between assays and sample concentrations.(Σ x)2 /n b= y. Linearity It may be demonstrated directly on the drug substance (by dilution of a standard stock solution) and/or separate weightings' of synthetic mixtures of the drug product components. b intercept m= Σ xy . the test data may need to be subjected to a mathematical transformation prior to the regression analysis. Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. test results should be evaluated by appropriate statistical methods. If there is a linear relationship. using the proposed procedure. Y = mX + b where Y is test result.

1 – 0.For the establishment of linearity.8 mg% and the same concentration of salicylic acid 2 mg % at 274 nm . a minimum of 5 concentrations is .recommended • Chromatogram of different concentration of norfloxacin from 0.

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

Range • The range of an analytical method is the interval between the upper and lower levels of the analyte which could be determined with an acceptable degree of linearity. up to 90%. if the specifications for a controlled released product cover a region from 20%. based on the nature of the dosage form (e. metered dose inhalers). e. • • For the determination of an impurity: from the reporting level of an impurity 1 to 120% of the specification . covering a minimum of 70 to 130 percent of the test concentration. the validated range would be 0-110% of the label claim.g. is justified. after 1 hour. accuracy and precision. unless a wider more appropriate range..g. The following minimum specified ranges should be considered: for the assay of a drug substance or a finished (drug) product: normally from 80 to 120 percent of the test concentration. after 24 hours.• 3. for content uniformity. for dissolution testing: +/-20 % over the specified range..

It expresses the correctness of the result. although it has larger absolute error. Example Atomic absorption analysis of As+3 and pb+2 in a sample yield the following results As+3 = 600 µg.100 .100 E rel As+3 = 5/600 x100 =0.3/9 x100 = 3.3 % From the aforementioned. it is obvious that As+3 results is more we use relative errors.83% E rel pb+2 = 0.3 µg. ACCURACY Accuracy is the degree of agreement between the experimental result and the true value. Absolute error (d) The difference between the analytical result and the true value d= x-µ where x observed value. Accuracy should be established across the specified range of the analytical procedure. d = From absolute error As+3 results is less accurate than pb+2 results but is not true as we see from relative error. C. µ true value Absolute error has no significance when separated from true or observed value .100 = amount found/ amount claimed or calculated .ml-1 d = 5 µg. ml-1 +2 pb = 9 µg. or most probable value. Measures to express accuracy A. Recovery % (Relative accuracy) = x/µ .• • • • • • • • • • • • • • • • • • 4.Relative error (E rel) E rel = d/µ .

it may be acceptable either to add known quantities of the analyte to the drug product or to compare the results obtained from a second. 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 mean and the accepted true value together with the confidence intervals. reference material).. the accuracy of which is stated and/or defined For Impurities (Quantization) Accuracy should be assessed on samples (drug substance/drug product) spiked with known amounts of impurities.g. weight/weight or area percent. Comparison of the results of the proposed analytical procedure with those of a second well-characterized procedure. For Drug Product a) Application of the analytical procedure to synthetic mixtures of the drug product components to which known quantities of the drug substance to be analyzed have been added. in all cases with respect to the major analyte. It should be clear how the individual or total impurities are to be determined e. well characterized procedure.• • • Several methods of determining accuracy are available: For Drug Substance a) Application of an analytical procedure to an analyte of known purity (e. In cases where it is impossible to obtain samples of certain impurities and/or degradation products. • • • • • • • . the accuracy of which is stated and/or defined. Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range (e. it is considered acceptable to compare results obtained by an independent procedure.g. b) In cases where it is impossible to obtain samples of all drug product components. 3 concentrations /3 replicates each of the total analytical procedure).g.

Examples of Precision and Accuracy: Low Accuracy High Precision High Accuracy Low Precision High Accuracy High Precision .

and N is the number of replicate assays. PRECISION • Precision is the degree of agreement among a series of measurements of the same quantity. 68. If the results are normally distributed. • The standard deviation is a popular estimate of the error in an analysis because it has statistical significance whenever the results are normally distributed.3 percent of the results can be expected to fall within the range of plus or minus one standard deviation of the mean as a result of random error. is the average of the results.• 5. a. xi represents each of the individual analytical results. Most analytical results exhibit normal (Gaussian) behavior. following the characteristic bell-shaped curve. where Σ represents summation. it is a measure of the reproducibility of results rather than their correctness • Measures to express precision • standard deviation (s) • Is calculated by using equation 1. .

(a) 5 mg% and the same concentration of Salicylic acid. .Chromatogram of the same concentration of Norfloxacin. (d) 2 mg% id at 274 nm.

between which the results are statistically expected to fall a given percentage of the time.3 ± 5.12 µ = 15.1 n= 4 Calculate the confidence limit of the mean at probability90% and 99% (t =2.100 also called coefficient of variation C . X.841 at probability 99%) µ = 15. µ=x. 3 concentrations/3 replicates each) Or b) A minimum of 6 determinations at 100% of the test concentration.841 x 0. Recommended Data The standard deviation.g.353 at 90 % probability and.353 x 0..3 s= 0.= 15. Relative standard deviation S rel = s /x. • • • • • • • • • • • • • • • .• • b.1/√4 µ = 15. relative standard deviation (coefficient of variation) and confidence interval should be reported for each type of precision investigated. obtaining the following results.± ts\ √n We might use this to estimate the probability that the population mean (µ) lies within a certain region centered at xExample A pharmacist determined the % of vitamin C in Rino C Tablets.3 ± 0.29 Repeatability should be assessed using: a) A minimum of 9 determinations covering the specified range for the procedure (e.3 ± 0.3 ± 2. 5.1/√4 µ = 15.Confidence limits at a given probability level are values greater than and less than the average.

1. 6. s= the standard deviation of the response S = the slope of the calibration curve The slope S may be estimated from the calibration curve of the analyte. Based on Visual Evaluation 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 be reliably detected.2. 6. Based on Signal-to-Noise This approach can only be applied to analytical procedures which exhibit baseline noise. Detection Limit Detection limit is the smallest concentration or amount of substances which can be reported with a specified degree of certainty by a definite. A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit. complete analytical procedure.3s/ S Where. 6. Several approaches for determining the detection limit are possible. depending on whether the procedure is a non-instrumental or instrumental.3 Based on the Standard Deviation of the Response and the Slope The detection limit (DL) may be expressed as: DL = 3. • • • • • • • • • • .• • • • • 6. 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 establishing the minimum concentration at which the analyte can be reliably detected.

• The estimate of s may be carried out in a variety of ways. • • 6. .3. for example: • • 6.1 Based on the Standard Deviation of the Blank Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the standard deviation of these responses. the presentation of the relevant chromatograms is considered acceptable for justification. The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation.2 Based on the Calibration Curve A specific calibration curve should be studied using samples containing an analyte in the range of DL.3. • • 6. If DL is determined based on visual evaluation or based on signal to noise ratio.4 Recommended Data The detection limit and the method used for determining the detection limit should be presented.

A typical signal-to-noise ratio is 10:1 Based on the Standard Deviation of the Response and the Slope The quantization limit (QL) may be expressed as: QL = 10s/ S Where s= the standard deviation of the response S = the slope of the calibration curve The slope S may be estimated from the calibration curve of the analyte. Based on Signal-to-Noise Approach 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 by establishing the minimum concentration at which the analyte can be reliably quantified.• • • • 7. Quantization Limit the minimum level at which the analyte can be quantified with acceptable accuracy and precision. • • • • • • • .

containing an analyte in the range of QL. Recommended Data The quantization limit and the method used for determining the quantization limit should be presented. Based on the Calibration Curve A specific calibration curve should be studied using samples.• • • The estimate of s may be carried out in a variety of ways for example: Based on Standard Deviation of the Blank Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the standard deviation of these responses. • • • . The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation. The limit should be subsequently validated by the analysis of a suitable number of samples known to be near or prepared at the quantization limit.

g. resolution test) is established to ensure that the validity of the analytical procedure is maintained whenever used. • Examples of typical variations are: stability of analytical solutions. temperature. influence of variations in mobile phase composition. flow rate.• 8. . extraction time. In the case of liquid chromatography. • One consequence of the evaluation of robustness should be that a series of system suitability parameters (e. • In the case of gas-chromatography. It should show the reliability during its usage. different columns (different lots and/or suppliers). examples of typical variations are different columns (different lots and/or suppliers). flow rate. Robustness • Robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters. examples of typical variations are influence of variations of pH in a mobile phase. temperature..

Ruggedness Ruggedness of an analytical method is the degree of reproducibility of the results obtained by the analysis of the same sample under a variety of normal test conditions for example different laboratory. System suitability testing The tests are based on the concept that the equipment. different instruments. electronics. • 10. analytical operations and samples to be analyzed constitutes an integral system that can be evaluated as such. • Sensitivity Sensitivity is the change in measured value resulting from a concentration change of one unit. and different reagents. different analysts. .• 9.