Methods validation includes an assessment of the adequacy of the analytical procedure. Statistical analysis (e.g., linear regression analysis, relative standard deviation) of methods validation data is often used to demonstrate the validity of the method. The statistical procedures for the analysis of the validation data should be determined prior to the start of any validation study. The procedure followed, including the amount of data to collect and the criteria used in determining the acceptability of the analytical procedure, should be specified.
The raw methods validation data and statistical procedures used to analyze the raw data should be provided and discussed in the sections on analytical procedures and controls. All statistical procedures used in the analysis of the data should be based on sound principles and be suitable for evaluating the dataset.
Comparative studies are performed to evaluate intermediate precision (e.g., different equipment, analysts, days). Comparative studies are also used to evaluate between laboratory variability (i.e., reproducibility) when an analytical procedure is used in more than one laboratory or to compare and evaluate the precision and accuracy of two analytical procedures (e.g., regulatory analytical procedure and an alternative analytical procedure). When comparative studies are performed, homogeneous samples from the same batch should be used, if feasible. Comparative results should be statistically analyzed and discussed and any bias explained.
For information on statistical techniques used in making comparisons, as well as other general information on the interpretation and treatment of analytical data, appropriate literature or texts should be consulted (see references) .
IX. REVALIDATION When sponsors make changes in the analytical procedure, drug substance (e.g., route of synthesis), or drug product (e.g., composition), the changes may necessitate revalidation of the analytical procedures. Revalidation should be performed to ensure that the analytical procedure maintains its characteristics (e.g., specificity) and to demonstrate that the analytical procedure continues to ensure the identity, strength, quality, purity, and potency of the drug substance and drug product, and the bioavailability of the drug product. The degree of revalidation depends on the nature of the change. When a different regulatory analytical procedure is substituted (e.g., HPLC for titration), the new procedure should be validated (see section VII).
If during each use an analytical procedure can meet the established system suitability requirements only with repeated adjustments to the operating conditions stated in the analytical procedure, the analytical procedure should be reevaluated, amended, and revalidated, as appropriate.
FDA intends to provide guidance in the future on postapproval changes in analytical procedures.
X. METHODS VALIDATION PACKAGE: CONTENTS AND PROCESSING Part of the methods validation process may include FDA laboratory analysis to demonstrate that an analytical procedure is reproducible by laboratory testing. A methods validation package (see X.A) and samples (see X.B) will be needed for this process.
A. Methods Validation Package
The methods validation package will usually include information copied from pertinent sections of the application. To aid the review chemist, these copies should retain the original pagination of the application sections. For ANDA and NDA products, the archival copy and extra copies of the methods validation packages should be submitted with the application. For ANDAs and related supplemental applications, one archival copy and two extra copies of the methods validation package should be submitted. For NDAs and related supplemental applications, one archival copy and three extra copies should be submitted. For BLAs and PLAs, a separate methods validation package need not be submitted. Information similar to that specified here should be included in the BLA or PLA submission.
The list should include the lot number, identity (with chemical name and structure where required for clarity), package type and size, date of manufacture, and quantity of the samples.
2. Analytical Procedures
A detailed description of each of the analytical procedures listed in the specifications should be submitted. The description should be sufficient to allow the FDA laboratory analysts to perform the analytical procedure (see section VI).
3. Validation Data
Appropriate validation data to support the analytical procedures should be submitted. Individual values as well as summary tables should be provided. Representative instrument output and raw data and information regarding stress studies should be included (see section VII).
4. Results
The results obtained by the applicant for the submitted samples should be provided. Alternatively, COAs could be submitted. The dates of analysis should be stated.
5. Composition
The components and composition of the drug product should be provided.
6. Specifications
The specifications for the drug substance and the drug product should be included.
7. Material Safety Data Sheets
The applicant should include material safety data sheets (MSDSs) for all samples, standards, and reagents (29 CFR 1910.1200(g)). As appropriate, MSDSs should be provided for other materials used in the analytical procedures listed in the methods validation package. In the case of toxic or hazardous materials, MSDSs should be posted on the outside of the package to facilitate safe handling.
On request from CDER, an NDA or ANDA applicant must submit samples of drug product, drug substance, noncompendial reference standards, and blanks, so that the suitability of the applicant=s drug substance and drug product analytical procedures can be evaluated by FDA laboratories (21 CFR 314.50(e) and 314.94(a)(10)). For BLAs and PLAs, representative samples of the product must be submitted, and summaries of the results of tests performed on the lots represented by the submitted sample must be provided (21 CFR 601.2(a) and 601.2(c)(1)(vi)).
For CDER products, the number of sets of samples that should be submitted for methods validation will be identified in the instructions forwarded to the applicant by the FDA laboratory. In general, the quantity of samples in each set should be double the amount needed to carry out the testing as performed by the applicant. Along with the drug substance and the drug product samples, the applicant should submit internal standards, non-USP reference standards, samples of impurities, degradation products, and unusual reagents. A set of samples will be shipped to each assigned laboratory.
For biological products, CBER should be consulted on the submission of samples and supporting materials.
Unless specified differently by the reviewer, samples from any batch, preferably samples from an aged batch, may be selected for NDAs and NDA supplemental applications. The submitted drug product samples should be from a batch made with the proposed market formulation. For ANDAs and appropriate supplements, a sample of the finished product from a batch being used to support approval of the submission should be used. If a sample is selected from a batch not described in the application, an amendment containing a copy of the batch record and certificate of analysis should be provided to the ANDA. For supplements that do not require submission and review of an exhibit batch record and associated data, any commercial batch may be submitted. For biological products, samples from several consecutively manufactured batches should be submitted.
The drug product should be supplied in its original packaging. Bulk substances (e.g., drug substances, impurities, excipients) should be stored in opaque nonreactive containers. To prevent breakage during shipping, the samples should be adequately packaged in a sturdy container. Samples shipped from outside the United States should contain the appropriate customs forms to reduce delay in delivery.
If special storage precautions (e.g., freezing, use of an inert gas blanket) are required to protect sample integrity, arrangements should be made in advance with the validating laboratory for scheduled direct delivery. If a sample is toxic or potentially hazardous, the container should be prominently labeled with an appropriate warning and precautionary handling instructions.
In the sections of the application on analytical procedures and controls, the applicant should provide a name, address, telephone number, and facsimile number so that samples can be requested. If this information is not provided, the contact person and address listed in the NDA, ANDA, BLA, or PLA submission will be used.
The methods validation packages should be compiled and submitted with the NDA or ANDA submission. For BLAs and PLAs, a separate methods validation package need not be submitted. When an FDA laboratory contacts the applicant for samples, the applicant should provide FDA laboratories with the samples within 10 working days. With the exception of sample delivery arrangements, all communications concerning validation at the FDA laboratories should be made through or with the knowledge of the review chemist for CDER applications, or the BLA/PLA committee chair for CBER applications.
The review chemist will review the application to determine that the analytical procedures are adequate to ensure the identity, strength, quality, purity, and potency of the drug substance and/or drug product. Any changes in the methods resulting from the review of the application may require resubmission of the methods validation package. The review chemist, in coordination with the appropriate FDA laboratories, will decide which analytical procedures are to be validated. Comments from the FDA laboratories, if any, will be forwarded by the review chemist to the applicant on completion of the studies by the laboratories.
An FDA laboratory will contact applicants with instructions on the submission of samples and the addresses to which samples should be mailed. The laboratory will test the samples according to the submitted analytical procedures to determine whether the analytical procedures are acceptable for quality control and suitable for regulatory purposes. Results and comments will be forwarded to the review chemist on completion of the studies.
4. Investigator
The investigator inspects the analytical laboratory testing sites where the release and stability testing are performed to ensure that the analytical procedures are performed in compliance with CGMP/GLP.
XI. METHODOLOGY Sections II through IX provide general information on the submission of analytical procedures and methods validation information, including validation characteristics. Additional information on certain methodologies is provided below.
The widespread use of HPLC analytical procedures and the multitude of commercial sources of columns and packings frequently have created problems in assessing comparability. Many of the following points may also apply to other chromatographic analytical procedures.
1. Column
The following characteristics are useful for defining a particular column and, if known, should be included in the analytical procedure description. If method development has indicated that columns from only one commercial source are suitable, this information should be included as part of the analytical procedure. If more than one column is suitable, a listing of columns found to be equivalent should be included.
Each analytical procedure submitted should include an appropriate number of system suitability tests defining the critical characteristics of that system. Criteria for all system suitability testing should be provided. The system suitability tests listed below are defined in CDER=s reviewer guidance on Validation of Chromatographic Methods (November 1994).
1. Tailing factor
2. Relative retention
3. Resolution
4. Relative standard deviation (RSD)
5. Capacity factor
6. Number of theoretical plates
2.系统适应性研究
CDER的评审官指南：色谱方法的验证(1994年11月) 中对如下这些系统适应性实验进行了定义：
1.拖尾因子
2. 相对保留时间
3.分离度
4.相对标准偏差
5.容量因子
6.理论塔板数
The RSD is normally performed at the beginning of the run. However, for assays with lengthy run times or as otherwise justified by the applicant, the reported average may be taken from injections at the beginning and end of the run, or at the beginning, middle, and end of the run.
If an internal standard is used, the minimum acceptable resolution between theinternal standard and one or more active ingredients should be specified. If theanalytical procedure is used to control the level of impurities, the minimum resolution between the active ingredient and the closest eluting impurity, or the two peaks eluting closest to each other, should be given.
The sequence of injection of blanks, system suitability standards, other standards, and samples should be defined. Flow rates, temperatures, and gradients should be described.Complete details should be provided for the preparation of the mobile phase,including the order of addition of the reagents and the methods of degassing andfiltration. The effect of adjustments in mobile phase composition on retention times should be included in the analytical procedure. The rationale for the use ofprecolumns and/or guard columns should be provided and justified. Any specialrequirements, such as the use of inert tubing or injection valves, should be specified.
At a minimum, the following parameters should be included in the description of a gc procedure. Additional parameters should be specified if required by the analytical procedure. If method development has indicated that columns from only one commercial source are suitable, this information should be included as part of the analytical procedure. If more than one column is suitable, a listing of columns found to be equivalent should be included.
---Column material (e.g., silica, glass, stainless steel)
--- Column conditioning procedure
2. Operating Parameters
---Gases: purity, flow rate, pressure
---Temperatures: column, injector, detector (including temperature program, if used)
---Injection (e.g., split, splitless, on-column)
---Detector
---Typical retention time and total run time
1.色谱柱
色谱柱尺寸：柱长，内外径
固定相
柱填充料(比如：硅酸，玻璃，不锈钢)
柱调节程序
2.操作参数
载气：纯度，流速，压力
温度：色谱柱，进样器，检测器(包括升温程序，如用到的话)
进样：(比如：分流，不分流，柱头进样(On-column injection)
检测限 典型保留时间和总运行时间。
3. System Suitability Testing
Appropriate system suitability criteria should be defined and included in all analytical procedures.
If an internal standard is used, the minimum acceptable resolution between the internal standard and one or more active ingredient should be specified. If the analytical procedure is used to control the level of impurities, the minimum resolution between the active ingredient and the closest eluting impurity, or the two peaks eluting closest to each other, should be given.
The RSD is normally performed at the beginning of the run. However, for assays with lengthy run times or as otherwise justified by the applicant, the reported average may be taken from injections at the beginning and end of the run, or beginning, middle, and end of the run.
C. Spectrophotometry, Spectroscopy, Spectrometry and Related Physical Methodologies
These analytical procedures include, but are not limited to, IR spectrophotometry, near IR spectrophotometry (NIR), UV/visible spectrophotometry (UV/Vis), atomic emission and atomic absorption, NMR, Raman spectroscopy, MS, and XRD.
Spectrometric analytical procedures may not be stability-indicating. The bias of the analytical procedure should be evaluated by comparing it with a chromatographic procedure, where appropriate. When manually operated equipment is used, the description of the analytical procedure should include an acceptance criterion for the amount of time that may elapse between sampling and reading. Appropriate system suitability and/or calibration testing is recommended. Validation criteria should include specificity (demonstrating no interference of placebo), linearity, repeatability, intermediate precision, and robustness.
At a minimum, the parameters listed below should be specified for a capillary electrophoretic analytical procedure. Additional parameters may be included as required by the procedure.
If method development has indicated that capillaries from only one commercial source are suitable, this information should be included as part of the analytical procedure. If more than one capillary is suitable, a listing of capillaries found to be equivalent should be included.
1. Capillary
--Capillary dimensions: length, length to detector, internal diameter, external diameter
-- Capillary preparation procedure: procedure to be followed before the first use, before the first run of the day, before each run (e.g., flush with 100 millimolar sodium hydroxide, flush with running buffer)
-- Running buffer: composition, including a detailed preparation procedure with the order of addition of the components
Each analytical procedure should include the appropriate system suitability tests defining the critical characteristics of that system. Other parameters may be included at the discretion of the applicant.
If an internal standard is used, the minimum acceptable resolution between the internal standard and one or more active ingredient should be specified. If the analytical procedure is used to control the level of impurities, the minimum resolution between the active ingredient and the closest eluting impurity, or the two peaks eluting closest to each other, should be given.
Optical rotation is used for the measurement of stereochemical purity. Visual polarimeters rely on a monochromatic source, which traditionally was sodium D, but has expanded to virtually any wavelength.
If measurements are to be made at a wavelength other than sodium D, an explanation for selecting the wavelength should be given, along with a comparison of the specific rotation at sodium D and the wavelength to be used. Circular dichroism (CD) spectra may suffice for this purpose. In addition to the provisions of USP <781>, procedures for measurement of specific rotation should include the solvent, concentration, and, for aqueous solutions, the pH to which the solution should be adjusted. The conditions and equipment should be shown to be suitable to confirm the stereochemical identity of a racemate or an enantiomer.
The enantiomeric purity can be expressed as enantiomeric excess (e.e.), using the following formula as an example:
where [M] and [m] are the concentrations of the major and minor enantiomers, respectively. This yields values of zero for a racemate and 100 percent for a pure enantiomer. An intermediate concentration gives intermediate values; for example, 97:3 would give an e.e. of 94 percent.
Appropriate system suitability and/or calibration testing is recommended. Validation criteria should include specificity, and intermediate precision. e.e. = 100% * {{M} - [m]}/{[M] + [m]}
Particle size analysis is an important element for quality control and regulatory evaluation of certain drug substances and drug products. The normal concepts of validation may differ for particle size methodologies as compared to other analytical methodologies such as HPLC.However, a standard mixture may be used for calibration.
Particle size evaluation can include characteristics of size, morphology, surface, and population of particles. The following parameters are useful for describing particle size analysis for characterization of drug substances and drug products.
To ensure proper instrument operation, the system should be calibrated according to the manufacturer's and/or the laboratory's specification, as appropriate.
The methods validation usually involves evaluation of intermediate precision and robustness. Assurance should be provided that the data generated are reproducible and control the product's quality. See additional information in sections V and VII.
The equipment used for dissolution is covered by USP <711> or USP <724>. The dissolution procedure description and validation should include the following.
A brief discussion of the reasons for selecting the medium.
2. Procedure
A dissolution test consists of a dissolution procedure and method of analysis (automated on-line analysis or manual sampling followed by HPLC analysis). The written procedure should cover the following items:
-- Apparatus
-- Preparation of standard
-- Preparation of sample
-- Method of analysis (e.g., UV, HPLC)
-- Sampling procedure (e.g., intervals, filtration, handling of samples, dilutions)
-- Calculations
-- Acceptance criteria
Regardless of the method of analysis, system suitability criteria should be described.Blank and standard solution spectra or chromatograms should be included.
Both the dissolution procedure and the method of analysis should be validated.
The time needed for the completion of the sample analysis should be stated in the procedure. Data should be submitted to support the stability of the dissolution sample during the procedure. If filters are used on-line or during sample preparation, appropriate recovery studies should be performed and documented and any bias should be addressed.
FDA encourages the development and use of the most appropriate instrumentation. However, the use of rare or exotic systems not only places an undue burden on the regulatory laboratory, but also may delay the validation process. When noncommercial instrumentation is used, the instrumentation should be capable of being constructed from commercially available components at a reasonable cost, if possible. For unique methodologies or instrumentation requiring contract fabrication, the applicant's cooperation with the FDA laboratories in helping facilitate duplication of the analytical procedure is important. In addition to design and equipment specifications, complete performance assessment procedures should be provided. Such systems may be found suitable for regulatory use.
The use of automated analytical procedures, although desirable for control testing, may lead to delay in regulatory methods validation because FDA laboratories have to assemble and validate the system before running samples. To avoid this delay, applicants should demonstrate the equivalence of a manual procedure to the automated procedure based on the same principle whenever possible.
The information relating to analytical procedures and methods validation that should be submitted in NDAs, ANDAs, BLAs, and PLAs is identified below with a cross-reference to the section of this guidance that provides recommendations and/or discussion on the topics.
Information that should be included in the analytical procedures and controls sections
1. Reference standard information Section IV
· Analytical procedures Section III, VI
· Validation data Section VII
· Stress studies Section VII.A.2.c
· Instrument output/raw data for impurities Section VII.A.2.b
Information that should be included in the methods validation package5
· Contents of the MV Package Section XI
· Representative instrument output/data for stress studies Section VII.A.2.c
·Representative instrument output and raw data for initialand oldest sample of a batch Section VII.A.2.b
分析方法验证中所需包括的资料：
分析方法验证的内容，第XI章
强降解实验的代表性仪器输出/原始资料，第VII.A.2.C章
某一批次最初样品和最老样品的代表性仪器输出/原始资料，第VII.A.2.b章
Information that should be included in the stability section
-- Stress study designs and results Section VII.A.2.b
-- Reference (volume and page number of submission)to instrument output and raw data submitted to the sectiondedicated to analytical procedures and controls Section VII.A 2.c
稳定性章节中所需包括的资料：
强降解实验的实验设计和实验结果，第VII.A.2.b章
参考分析方法和控制章节中所递交的的代表性仪器输出/原始资料，第VII.A.2.C章
ATTACHMENT B
METHODS VALIDATION PROBLEMS AND DELAY
Listed below are examples of common problems that can delay successful validation.
-- Failure to provide a sample of a critical impurity, degradation product, internal standard, or novel reagent
-- Failure to submit well-characterized reference standards for noncompendial drugs
-- Failure to provide sufficient detail or use of unacceptable analytical procedures. For example:
附录B
分析方法验证的问题和延误
下文举列说明了些会延误成功验证的常见问题。
--未能提供关键杂质，降解物，内标物或新试剂的样品。
--未能提供非药典药物的已界定标准品。
--未能提供分析方法的详细描述，或使用和了不可接受的分析方法。比如：
--Use of arbitrary arithmetic corrections
--Failure to provide system suitability tests
--Differing content uniformity and assay analytical procedures without showing
--equivalence factors for defining corrections as required by the current USP chapter
<905> - Uniformity of Dosage Units
-- Failure to submit complete or legible data. For example:
-- Failure to label instrument output to indicate sample identity
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