spectroscopic method may involve evaluation of Category Ior Category II Analytical Performance Characteristics, depend-ing on the method requirements. Qualitative physical prop-erty measurements, such as particle size, surface area, bulkand tapped density, which could impact performance char-acteristics, often best fit in Category III. Category IV AnalyticalPerformance Characteristics usually applies to validation ofqualitative identification spectroscopic methods. However,the various techniques may be used for different purposes,and the specific use of the method and characteristics of thematerial being analyzed should be considered when defini-tively applying a category to a particular type of method.

The validity of an analytical procedure can be verified onlyby laboratory studies. Therefore, documentation of the suc-cessful completion of such studies is a basic requirement fordetermining whether a procedure is suitable for its intendedapplication(s). Current compendial procedures are also sub-ject to regulations that require demonstration of suitabilityunder actual conditions of use (see Verification of CompendialProcedures 1226 for principles relative to the verification ofcompendial procedures). Appropriate documentationshould accompany any proposal for new or revised compen-dial analytical procedures.

1226 VERIFICATION OFCOMPENDIAL PROCEDURES

The intent of this general information chapter is to pro-vide general information on the verification of compendialprocedures that are being performed for the first time toyield acceptable results utilizing the personnel, equipment,and reagents available. This chapter is not intended for ret-roactive application to already successfully established labo-ratory procedures. The chapter Validation of Compendial Pro-cedures 1225 provides general information on characteris-tics that should be considered for various test categories andon the documentation that should accompany analyticalprocedures submitted for inclusion in USPNF. Verificationconsists of assessing selected analytical performance charac-teristics, such as those that are described in chapter 1225,to generate appropriate, relevant data rather than repeatingthe validation process.

Users of compendial analytical procedures are not re-quired to validate these procedures when first used in theirlaboratories, but documented evidence of suitability shouldbe established under actual conditions of use. In the UnitedStates, this requirement is established in 21 CFR 211.194(a)(2) of the current Good Manufacturing Practice regulations,which states that the suitability of all testing methods usedshall be verified under actual conditions of use.

Verification of microbiological procedures is not coveredin this chapter because it is covered in USP general testchapters Antimicrobial Effectiveness Testing 51, Microbiologi-cal Examination of Nonsterile Products: Microbial EnumerationTests 61, Microbiological Examination of Nonsterile Products:Tests for Specified Microorganisms 62, Sterility Tests 71,

and in general information chapter Validation of MicrobialRecovery from Pharmacopeial Articles 1227.

VERIFICATION PROCESS

The verification process for compendial test procedures isthe assessment of whether the procedure can be used for itsintended purpose, under the actual conditions of use for aspecified drug substance and/or drug product matrix.

Users should have the appropriate experience, knowl-edge, and training to understand and be able to performthe compendial procedures as written. Verification shouldbe conducted by the user such that the results will provideconfidence that the compendial procedure will perform suit-ably as intended.

If the verification of the compendial procedure is not suc-cessful, and assistance from USP staff has not resolved theproblem, it may be concluded that the procedure may notbe suitable for use with the article being tested in that labo-ratory. It may then be necessary to develop and validate analternate procedure as allowed in the General Notices. Thealternate procedure may be submitted to USP, along withthe appropriate data, to support a proposal for inclusion orreplacement of the current compendial procedure.

VERIFICATION REQUIREMENTS

Verification requirements should be based on an assess-ment of the complexity of both the procedure and the ma-terial to which the procedure is applied. Although completerevalidation of a compendial method is not required to veri-fy the suitability of a procedure under actual conditions ofuse, some of the analytical performance characteristics listedin chapter 1225, Table 2, may be used for the verificationprocess. Only those characteristics that are considered to beappropriate for the verification of the particular procedureneed to be evaluated. The process of assessing the suitabilityof a compendial analytical test procedure under the condi-tions of actual use may or may not require actual laboratoryperformance of each analytical performance characteristic.The degree and extent of the verification process may de-pend on the level of training and experience of the user, onthe type of procedure and its associated equipment or in-strumentation, on the specific procedural steps, and onwhich article(s) are being tested.

Verification should assess whether the compendial proce-dure is suitable for the drug substance and/or the drugproduct matrix, taking into account the drug substance'ssynthetic route, the method of manufacture for the drugproduct, or both, if applicable. Verification should includean assessment of elements such as the effect of the matrixon the recovery of impurities and drug substances from thedrug product matrix, as well as the suitability of chromato-graphic conditions and column, the appropriateness of de-tector signal response, etc.

As an example, an assessment of specificity is a key pa-rameter in verifying that a compendial procedure is suitablefor use in assaying drug substances and drug products. Forinstance, acceptable specificity for a chromatographic meth-od may be verified by conformance with system suitabilityresolution requirements (if specified in the procedure). How-

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ever, drug substances from different suppliers may have dif-ferent impurity profiles that are not addressed by the com-pendial test procedure. Similarly, the excipients in a drugproduct can vary widely among manufacturers and mayhave the potential to directly interfere with the procedure orcause the formation of impurities that are not addressed bythe compendial procedure. In addition, drug products con-taining different excipients, antioxidants, buffers, or contain-er extractives may affect the recovery of the drug substancefrom the matrix. In these cases, a more thorough assess-ment of the matrix effects may be required to demonstratesuitability of the procedure for the particular drug substanceor product. Other analytical performance characteristicssuch as an assessment of the limit of detection or quantita-tion and precision for impurities procedures may be usefulto demonstrate the suitability of the compendial procedureunder actual conditions of use.

Verification is not required for basic compendial test pro-cedures that are routinely performed unless there is an indi-cation that the compendial procedure is not appropriate forthe article under test. Examples of basic compendial proce-dures include, but are not limited to, loss on drying, residueon ignition, various wet chemical procedures such as acidvalue, and simple instrumental determinations such as pHmeasurements. However, for the application of already es-tablished routine procedures to compendial articles testedfor the first time, it is recommended that consideration begiven to any new or different sample handling or solutionpreparation requirements.

1227 VALIDATION OFMICROBIAL RECOVERY FROMPHARMACOPEIAL ARTICLES

This chapter provides guidelines for the validation ofmethods for the estimation of the number of viable microor-ganisms, for the detection of indicators or objectionable mi-croorganisms, for the validation of microbiological methodsused in antimicrobial effectiveness testing, and for the sterili-ty testing of Pharmacopeial articles. It is generally under-stood that if a product possesses antimicrobial propertiesbecause of the presence of a specific preservative or becauseof its formulation, this antimicrobial property must be neu-tralized to recover viable microorganisms. This neutraliza-tion may be achieved by the use of a specific neutralizer, bydilution, by a combination of washing and dilution, or byany combination of these methods.

The tests under Antimicrobial Effectiveness Testing 51,Sterility Tests 71, and Microbial Enumeration Tests 61 andTests for Specified Microorganisms 62 require the validationof recovery methods. To ensure that the results of the testsare credible, neutralization of antimicrobial properties of thetest solution is required before estimating the number ofviable microorganisms.

INFLUENTIAL FACTORS

Several factors affect the measurement of a test solution'santimicrobial activity, and these must be considered in thevalidation design. They include the nature of the microor-ganisms used as challenge organisms, the preparation of theinoculum of challenge organisms, the specific conditions ofthe test, and the conditions of recovery. These factors alsoaffect the validation of recovery methods for aqueous ornonaqueous products, irrespective of their antimicrobialproperties; thus, all test methods should be validated withthese factors in mind.

The nature of the challenge microorganism exerts astrong effect upon the response to the antimicrobial agent,and so upon the neutralization required for recovery. Repre-sented among these organisms in compendial tests areGram-positive bacteria, Gram-negative bacteria, yeasts, andmolds. Each organism to be used in the test must be inclu-ded in the validation.

The preparation of the inoculum of challenge microor-ganisms also affects the testing of products having antimi-crobial properties. The growth and preparation of the chal-lenge organism determines the physiological state of thecell. This state has a direct influence on the results of anytest of antimicrobial efficacy. Microbial tests do not use indi-vidual cells; rather, populations of cells are harvested forstudy. The data generated from these studies are less varia-ble if the cell populations are homogeneous. Liquid culturesor confluent growths on solid medium are best suited for re-producible culture preparation. The conditions of organismpreparation and storage must be standardized for the neu-tralizer evaluation and should reflect the conditions of theantimicrobial assay.

The specific conditions of the test, including buffers used,water, light conditions, and temperature, must be repro-duced in the validation study. All test conditions also shouldbe standardized and performed in the validation study ex-actly as performed in the test.

The conditions of microbial recovery are among the mostcrucial in accurately estimating the number of microorgan-isms present in a test solution. The first consideration is therecovery medium used to support the growth of survivors.This concern is discussed in detail below. The second con-sideration is the incubation conditions. Optimal conditionsfor growth must be present to ensure complete growth andreproducible results.

METHODS OF NEUTRALIZINGANTIMICROBIAL PROPERTIES

Three common methods are used to neutralize antimicro-bial properties of a product: (1) chemical inhibition, (2) dilu-tion, and (3) filtration and washing.

Chemical Inhibition

Table 1 shows known neutralizers for a variety of chemicalantimicrobial agents and the reported toxicity of somechemical neutralizers to specific microorganisms. However,despite potential toxicity, the convenience and quick actionof chemical inhibitors encourage their use. Chemical inhibi-

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