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`1225 VALIDATION OF COMPENDIAL METHODS
`
`Test procedures for assessment of the quality levels of pharmaceutical products are subject to various requirements.
`According to Section 501 of the Federal Food, Drug, and Cosmetic Act, assays and specifications in monographs of the
`United States Pharmacopeia and the National Formulary constitute legal standards. The Current Good Manufacturing
`Practice regulations [21 CFR 211.194(a)] require that test methods, which are used for assessing compliance of
`pharmaceutical products with established specifications, must meet proper standards of accuracy and reliability. Also,
`according to these regulations [21 CFR 211.194(a)(2)], users of analytical methods described in the USP and the NF are
`not required to validate accuracy and reliability of these methods, but merely verify their suitability under actual conditions
`of use. Recognizing the legal status of USP and NF standards, it is essential, therefore, that proposals for adoption of new
`or revised compendial analytical methods be supported by sufficient laboratory data to document their validity.
`
`The text of this information chapter harmonizes, to the extent possible, with the Tripartite International Conference on
`Harmonization (ICH) documents Validation of Analytical Procedures and the Methodology extension text, which are
`concerned with analytical procedures included as part of registration applications submitted within the EC, Japan, and the
`USA. Some aspects (dissolution, drug release), which form part of this chapter, are dealt with only in passing in the ICH
`documents and are to be discussed in the future. Complete harmonization has not been possible, in part because of
`different uses of terminology. For example, the ICH use of “procedure” presents difficulty, because this term has a specific
`and different use throughout the USP–NF.
`
`SUBMISSIONS TO THE COMPENDIA
`
`Submissions to the compendia for new or revised analytical methods should contain sufficient information to enable
`members of the USP Committee of Revision to evaluate the relative merit of proposed procedures. In most cases,
`evaluations involve assessment of the clarity and completeness of the description of the analytical methods, determination
`of the need for the methods, and documentation that they have been appropriately validated. Information may vary
`depending upon the type of method involved. However, in most cases a submission will consist of the following sections.
`
`Rationale— This section should identify the need for the method and describe the capability of the specific method
`proposed and why it is preferred over other types of determinations. For revised procedures, a comparison should be
`provided of limitations of the current compendial method and advantages offered by the proposed method.
`
`Proposed Analytical Procedure— This section should contain a complete description of the analytical method sufficiently
`detailed to enable persons skilled in the art to replicate it. The write-up should include all important operational parameters
`and specific instructions such as preparation of reagents, performance of systems suitability tests, description of blanks
`used, precautions, and explicit formulas for calculation of test results.
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`Data Elements— This section should provide thorough and complete documentation of the validation of the analytical
`method. It should include summaries of experimental data and calculations substantiating each of the applicable analytical
`performance characteristics. These characteristics are described in the following section.
`
`VALIDATION
`
`Validation of an analytical method is the process that establishes, by laboratory studies, that the performance
`characteristics of the method meet the requirements for the intended analytical applications. Typical analytical performance
`characteristics that should be considered in the validation of the types of methods described in this document are listed in
`Table 1. Since opinions may differ with respect to terminology and use, each of the performance characteristics is defined
`in the next section of this chapter along with a delineation of a typical method or methods by which it may be measured.
`
`Table 1. Typical Analytical Characteristics Used in Method Validation
`Accuracy
`Precision
`Specificity
`Detection limit
`Quantitation limit
`Linearity
`Range
`Ruggedness
`
`In the case of compendial methods, revalidation may be necessary in the following cases: a submission to the USP of a
`revised analytical method; or the use of an established general method with a new product or raw material (see below
`under Data Elements Required for Assay Validation).
`
`The ICH documents give guidance on the necessity for revalidation in the following circumstances: changes in the
`synthesis of the drug substance, changes in the composition of the drug product, and changes in the analytical procedure.
`
`ANALYTICAL PERFORMANCE CHARACTERISTICS
`
`Accuracy—
`
`Definition— The accuracy of an analytical method is the closeness of test results obtained by that method to the true value.
`The accuracy of an analytical method should be established across its range.
`
`Determination— In assay of a drug substance, accuracy may be determined by application of the analytical method to an
`analyte of known purity (e.g., a Reference Standard) or by comparison of the results of the method with those of a second,
`well-characterized method, the accuracy of which has been stated or defined.
`
`In assay of a drug in a formulated product, accuracy may be determined by application of the analytical method to synthetic
`mixtures of the drug product components to which known amounts of analyte have been added within the range of the
`method. If it is not possible to obtain samples of all drug product components, it may be acceptable either to add known
`quantities of the analyte to the drug product (i.e., to “spike”) or to compare results with those of a second, well-
`characterized method, the accuracy of which has been stated or defined.
`
`In quantitative analysis of impurities, accuracy should be assessed on samples (of drug substance or drug product) spiked
`with known amounts of impurities. Where it is not possible to obtain samples of certain impurities or degradation products,
`results should be compared with those obtained by an independent method. In the absence of other information, it may be
`necessary to calculate the amount of an impurity on the basis of comparison of its response to that of the drug substance;
`the ratio of the responses of equal amounts of the impurity and the drug substance (response factor) should be used if
`known.
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`Accuracy is calculated as the percentage of recovery by the assay of the known added amount of analyte in the sample, or
`as the difference between the mean and the accepted true value, together with confidence intervals.
`
`The ICH documents recommend that accuracy be assessed using a minimum of nine determinations over a minimum of
`three concentration levels, covering the specified range (i.e., three concentrations and three replicates of each
`concentration).
`
`Precision—
`
`Definition— The precision of an analytical method is the degree of agreement among individual test results when the
`method is applied repeatedly to multiple samplings of a homogeneous sample. The precision of an analytical method is
`usually expressed as the standard deviation or relative standard deviation (coefficient of variation) of a series of
`measurements. Precision may be a measure of either the degree of reproducibility or repeatability of the analytical method
`under normal operating conditions. In this context, reproducibility refers to the use of the analytical procedure in different
`laboratories, as in a collaborative study. Intermediate precision expresses within-laboratory variation, as on different days,
`or with different analysts or equipment within the same laboratory. Repeatability refers to the use of the analytical
`procedure within a laboratory over a short period of time using the same analyst with the same equipment. For most
`purposes, repeatability is the criterion of concern in USP analytical procedures, although reproducibility between
`laboratories or intermediate precision may well be considered during the standardization of a procedure before it is
`submitted to the Pharmacopeia.
`
`Determination— The precision of an analytical method is determined by assaying a sufficient number of aliquots of a
`homogeneous sample to be able to calculate statistically valid estimates of standard deviation or relative standard deviation
`(coefficient of variation). Assays in this context are independent analyses of samples that have been carried through the
`complete analytical procedure from sample preparation to final test result.
`
`The ICH documents recommend that repeatability should be assessed using a minimum of nine determinations covering
`the specified range for the procedure (i.e., three concentrations and three replicates of each concentration, or a minimum of
`six determinations at 100% of the test concentration).
`
`Specificity—
`
`Definition— The ICH documents define specificity as the ability to assess unequivocally the analyte in the presence of
`components that may be expected to be present, such as impurities, degradation products, and matrix components. Lack
`of specificity of an individual analytical procedure may be compensated for by other supporting analytical procedures.
`[NOTE—Other reputable international authorities (IUPAC, AOAC) have preferred the term “selectivity,” reserving “specificity”
`for procedures that are completely selective.] For the test or assay methods below, the above definition has the following
`implications:
`
`IDENTIFICATION TESTS— ensure the identity of the analyte.
`
`PURITY TESTS— ensure that all the analytical procedures performed allow an accurate statement of the content of impurities
`of an analyte (e.g., related substances test, heavy metals limit, organic volatile impurity limit).
`
`ASSAYS— provide an exact result, which allows an accurate statement on the content or potency of the analyte in a sample.
`
`Determination— In qualitative analyses (identification tests), the ability to select between compounds of closely related
`structure that are likely to be present should be demonstrated. This ability should be confirmed by obtaining positive results
`(perhaps by comparison to a known reference material) from samples containing the analyte, coupled with negative results
`from samples that do not contain the analyte, and by confirming that a positive response is not obtained from materials
`structurally similar to or closely related to the analyte.
`
`In an analytical procedure for impurities, specificity may be established by spiking the drug substance or product with
`appropriate levels of impurities and demonstrating that these impurities are determined with appropriate accuracy and
`precision.
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`In an assay, demonstration of specificity requires that it can be shown that the procedure is unaffected by the presence of
`impurities or excipients. In practice, this can be done by spiking the drug substance or product with appropriate levels of
`impurities or excipients and demonstrating that the assay result is unaffected by the presence of these extraneous
`materials.
`
`If impurity or degradation product standards are unavailable, specificity may be demonstrated by comparing the test results
`of samples containing impurities or degradation products to a second well-characterized procedure (e.g., a pharmacopeial
`or other validated procedure). These comparisons should include samples stored under relevant stress conditions (e.g.,
`light, heat, humidity, acid or base hydrolysis, oxidation). In an assay, the results should be compared; in chromatographic
`impurity tests, the impurity profiles should be compared.
`
`The ICH documents state that when chromatographic procedures are used, representative chromatograms should be
`presented to demonstrate the degree of selectivity, and peaks should be appropriately labeled. Peak purity tests (e.g.,
`using diode array or mass spectrometry) may be useful to show that the analyte chromatographic peak is not attributable to
`more than one component.
`
`Detection Limit—
`
`Definition— The detection limit is a characteristic of limit tests. It is the lowest amount of analyte in a sample that can be
`detected, but not necessarily quantitated, under the stated experimental conditions. Thus, limit tests merely substantiate
`that the amount of analyte is above or below a certain level. The detection limit is usually expressed as the concentration of
`analyte (e.g., percentage, parts per billion) in the sample.
`
`Determination— For noninstrumental methods, the detection limit is generally 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.
`
`For instrumental procedures, the same method may be used as for noninstrumental. In the case of methods submitted for
`consideration as official compendial methods, it is almost never necessary to determine the actual detection limit. Rather,
`the detection limit is shown to be sufficiently low by the analysis of samples with known concentrations of analyte above
`and below the required detection level. For example, if it is required to detect an impurity at the level of 0.1%, it should be
`demonstrated that the procedure will reliably detect the impurity at that level.
`
`In the case of instrumental analytical procedures that exhibit background noise, the ICH documents describe a common
`approach, which is to compare measured signals from samples with known low concentrations of analyte with those of
`blank samples. The minimum concentration at which the analyte can reliably be detected is established. Typically
`acceptable signal-to-noise ratios are 2:1 or 3:1. Other approaches depend on the determination of the slope of the
`calibration curve and the standard deviation of responses. Whatever method is used, the detection limit should be
`subsequently validated by the analysis of a suitable number of samples known to be near, or prepared at, the detection
`limit.
`
`Quantitation Limit—
`
`Definition— The quantitation limit is a characteristic of quantitative assays for low levels of compounds in sample matrices,
`such as impurities in bulk drug substances and degradation products in finished pharmaceuticals. It is the lowest amount of
`analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental
`conditions. The quantitation limit is expressed as the concentration of analyte (e.g., percentage, parts per billion) in the
`sample.
`
`Determination— For noninstrumental methods, the quantitation limit is generally determined by the analysis of samples
`with known concentrations of analyte and by establishing the minimum level at which the analyte can be determined with
`acceptable accuracy and precision.
`
`For instrumental procedures, the same method may be used as for noninstrumental. In the case of methods submitted for
`consideration as official compendial methods, it is almost never necessary to determine the actual quantitation limit. Rather,
`the quantitation limit is shown to be sufficiently low by the analysis of samples with known concentrations of analyte above
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`and below the quantitation level. For example, if it is required to assay an analyte at the level of 0.1 mg per tablet, it should
`be demonstrated that the method will reliably quantitate the analyte at that level.
`
`In the case of instrumental analytical methods that exhibit background noise, the ICH documents describe a common
`approach, which is to compare measured signals from samples with known low concentrations of analyte with those of
`blank samples. The minimum concentration at which the analyte can reliably be quantified is established. A typically
`acceptable signal-to-noise ratio is 10:1. Other approaches depend on the determination of the slope of the calibration curve
`and the standard deviation of responses. Whatever method is used, the quantitation limit should be subsequently validated
`by the analysis of a suitable number of samples known to be near, or prepared at, the quantitation limit.
`
`Linearity and Range—
`
`Definition of Linearity— The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-
`defined mathematical transformation, proportional to the concentration of analyte in samples within a given range.
`
`Definition of Range— The range of an analytical method is the interval between the upper and lower levels of analyte
`(including these levels) that has been demonstrated to be determined with a suitable level of precision, accuracy, and
`linearity using the method as written. The range is normally expressed in the same units as test results (e.g., percent, parts
`per million) obtained by the analytical method.
`
`Determination of Linearity and Range— Linearity should be established across the range of the analytical procedure. It
`should be established initially by visual examination of a plot of signals as a function of analyte concentration of content. If
`there appears to be a linear relationship, test results should be established by appropriate statistical methods (e.g., by
`calculation of a regression line by the method of least squares). In some cases, to obtain linearity between the response of
`an analyte and its concentration, the test data may have to be subjected to a mathematical transformation. Data from the
`regression line itself may be helpful for providing mathematical estimates of the degree of linearity. The correlation
`coefficient, y-intercept, slope of the regression line, and residual sum of squares should be submitted.
`
`The range of the method is validated by verifying that the analytical method provides acceptable precision, accuracy, and
`linearity when applied to samples containing analyte at the extremes of the range as well as within the range.
`
`ICH recommends that, for the establishment of linearity, a minimum of five concentrations normally be used. It is also
`recommended that the following minimum specified ranges should be considered:
`
`ASSAY OF A DRUG SUBSTANCE (or a finished product): from 80% to 120% of the test concentration.
`
`DETERMINATION OF AN IMPURITY: from 50% to 120% of the specification.
`
`FOR CONTENT UNIFORMITY: a minimum of 70% to 130% of the test concentration, unless a wider or more appropriate range,
`based on the nature of the dosage form (e.g., metered-dose inhalers) is justified.
`
`FOR DISSOLUTION TESTING: ±20% over the specified range (e.g., if the specifications for a controlled-release product cover a
`region from 20% after 1 hour, and up to 90% after 24 hours, the validated range would be 0% to 110% of the label claim).
`
`Ruggedness—
`
`Definition— The ruggedness of an analytical method is the degree of reproducibility of test results obtained by the analysis
`of the same samples under a variety of conditions, such as different laboratories, analysts, instruments, lots of reagents,
`elapsed assay times, assay temperatures, or days. Ruggedness is normally expressed as the lack of influence on test
`results of operational and environmental variables of the analytical method. Ruggedness is a measure of reproducibility of
`test results under the variation in conditions normally expected from laboratory to laboratory and from analyst to analyst.
`
`Determination— The ruggedness of an analytical method is determined by analysis of aliquots from homogeneous lots in
`different laboratories, by different analysts, using operational and environmental conditions that may differ but are still within
`the specified parameters of the assay. The degree of reproducibility of test results is then determined as a function of the
`assay variables. This reproducibility may be compared to the precision of the assay under normal conditions to obtain a
`measure of the ruggedness of the analytical method.
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`Robustness—
`
`Definition— The robustness of an analytical method is a measure of its capacity to remain unaffected by small but
`deliberate variations in method parameters and provides an indication of its reliability during normal usage.
`
`System Suitability— If measurements are susceptible to variations in analytical conditions, these should be suitably
`controlled, or a precautionary statement should be included in the method. One consequence of the evaluation of
`ruggedness and robustness should be that a series of system suitability parameters is established to ensure that the
`validity of the analytical method is maintained whenever used. Typical variations are the stability of analytical solutions,
`equipment, and analysts. In liquid chromatography, typical variations are the pH of the mobile phase, the mobile phase
`composition, different lots or suppliers of columns, the temperature, and the flow rate. In the case of gas chromatography,
`typical variations are different lots or suppliers of columns, the temperature, and the flow rate.
`
`System suitability tests are based on the concept that the equipment, electronics, analytical operations, and samples to be
`analyzed constitute an integral system that can be evaluated as such. System suitability test parameters to be established
`for a particular method depend on the type of method being evaluated. They are especially important in the case of
`chromatographic methods, and submissions to the USP should make note of the requirements under the System Suitability
`section in the general test chapter Chromatography
`621 .
`
`DATA ELEMENTS REQUIRED FOR ASSAY VALIDATION
`
`Compendial assay procedures vary from highly exacting analytical determinations to subjective evaluation of attributes.
`Considering this variety of assays, it is only logical that different test methods require different validation schemes. This
`chapter covers only the most common categories of assays for which validation data should be required. These categories
`are as follows.
`
`Category I: Analytical methods for quantitation of major components of bulk drug substances or active ingredients
`(including preservatives) in finished pharmaceutical products.
`
`Category II: Analytical methods for determination of impurities in bulk drug substances or degradation compounds in
`finished pharmaceutical products. These methods include quantitative assays and limit tests.
`
`Category III: Analytical methods for determination of performance characteristics (e.g., dissolution, drug release).
`
`Category IV: Identification tests.
`
`For each assay category, different analytical information is needed. Listed in Table 2 are data elements normally required
`for each of the categories of assays.
`
`Table 2. Data Elements Required for Assay Validation
`Assay
`Category II
`
`Analytical
`Limit
`Assay
`Performance
`Tests
`Quantitative
`Category I
`Characteristics
`Yes
`Yes
`Accuracy
`*
`No
`Yes
`Yes
`Precision
`Yes
`Yes
`Yes
`Specificity
`Yes
`No
`No
`Detection limit
`No
`Yes
`No
`Quantitation limit
`No
`Yes
`Yes
`Linearity
`Yes
`Yes
`Range
`*
`* May be required, depending on the nature of the specific test.
`
`Assay
`Category III
`*
`Yes
`*
`*
`*
`*
`*
`
`Assay
`Category IV
`No
`No
`Yes
`No
`No
`No
`No
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`Already established general assays and tests (e.g., titrimetric method of water determination, bacterial endotoxins test)
`should be revalidated to verify their accuracy (and absence of possible interference) when used for a new product or raw
`material.
`
`The validity of an analytical method can be verified only by laboratory studies. Therefore, documentation of the successful
`completion of such studies is a basic requirement for determining whether a method is suitable for its intended applications.
`Appropriate documentation should accompany any proposal for new or revised compendial analytical procedures.
`
`Auxiliary Information— Staff Liaison : Horacio Pappa, Ph.D.
`Expert Committee : (GC05) General Chapters 05
`USP29–NF24 Page 3050
`Pharmacopeial Forum : Volume No. 31(2) Page 549
`Phone Number : 1-301-816-8319
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