USP-化学药物质量控制分析方法验证技术指导原则

1225VALIDATION OF COMPENDIAL PROCEDURES

Test procedures for assessment of the quality levels of pharmaceutical articles 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 articles 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 USP–NF are not required to validate the 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 procedures 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.

SUBMISSIONS TO THE COMPENDIA

Submissions to the compendia for new or revised analytical procedures should contain sufficient information to enable members of the USP Council of Experts and its Expert Committees 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 procedures, determination of the need for the procedures, 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 procedure and describe the capability of the specific procedure 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 procedure and advantages offered by the proposed procedure.

Proposed Analytical Procedure— This section should contain a complete description of the analytical procedure 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 system suitability tests, description of blanks used, precautions, and explicit formulas for calculation of test results.

Data Elements— This section should provide thorough and complete documentation of the validation of the analytical procedure. 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 procedure is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for the intended analytical applications. Typical analytical performance characteristics that should be considered in the validation of the types of procedures described in this document are listed in Table 1. Because 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. The definitions refer to ―test results.‖ The description of the analytical procedure should define what the test results for the procedure are. As noted

in ISO 5725-1 and 3534-1, a test result is ―the value of a characteristic obtained by carrying out a specified test method. The test method should specify that one or a number of individual measurements be made, and their average, or another appropriate function (such as the median or the standard deviation), be reported as the test result. It may also require standard corrections to be applied, such as correction of gas volumes to standard temperature and pressure. Thus, a test result can be a result calculated from several observed values. In the simple case, the test result is the observed value itself.‖ A test result al so can be, but need not be, the final, reportable value that would be compared to the acceptance criteria of a specification. Validation of physical property methods may involve the assessment of chemometric models. However, the typical analytical characteristics used in method validation can be applied to the methods derived from the use of the chemometric models.

The effects of processing conditions and potential for segregation of materials should be considered when obtaining a representative sample to be used for validation of procedures.

Table 1. Typical Analytical Characteristics

Used in Method Validation

Accuracy

Precision

Specificity

Detection Limit

Quantitation Limit

Linearity

Range

Robustness

In the case of compendial procedures, revalidation may be necessary in the following cases: a submission to the USP of a revised analytical procedure; or the use of an established general procedure with a new product or raw material (see below in Data Elements Required for 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.

Chapter 1225is intended to provide information that is appropriate to validate a wide range of compendial analytical procedures. The validation of compendial procedures may use some or all of the suggested typical analytical characteristics used in method validation as outlined in Table 1 and categorized by type of analytical method in Table 2. For some compendial procedures the fundamental principles of validation may extend beyond characteristics suggested in Chapter 1225. For these procedures the user is referred to the individual compendial chapter for those specific analytical validation characteristics and any specific validation requirements.

Analytical Performance Characteristics

accuracy

Definition— The accuracy of an analytical procedure is the closeness of test results obtained by that procedure to the true value. The accuracy of an analytical procedure should be established across its range. [A note on terminology: The definition of accuracy in 1225and ICH Q2 corresponds to unbiasedness only. In the International Vocabulary of Metrology (VIM) and documents of the International Organization for Standardization (ISO),

―accuracy‖ has a different meaning. In ISO, accur acy combines the concepts of unbiasedness (termed ―trueness‖) and precision.]

Determination— In the case of the assay of a drug substance, accuracy may be determined by application of the analytical procedure to an analyte of known purity (e.g., a Reference Standard) or by comparison of the results of the procedure with those of a second, well-characterized procedure, the accuracy of which has been stated or defined.

In the case of the assay of a drug in a formulated product, accuracy may be determined by application of the analytical procedure to synthetic mixtures of the drug product components to which known amounts of analyte have been added within the range of the procedure. 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 procedure, the accuracy of which has been stated or defined.

In the case of 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 procedure. In the absence of other information, it may be necessary to calculate the amount of an impurity based on 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 (relative response factor) should be used if known.

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 should 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).

Assessment of accuracy can be accomplished in a variety of ways, including evaluating the recovery of the analyte (percent recovery) across the range of the assay, or evaluating the linearity of the relationship between estimated and actual concentrations. The statistically preferred criterion is that the confidence interval for the slope be contained in an interval around 1.0, or alternatively, that the slope be close to 1.0. In either case, the interval or the definition of

closeness should be specified in the validation protocol. The acceptance criterion will depend on the assay and its variability and on the product. Setting an acceptance criterion based on the lack of statistical significance of the test of the null hypothesis that the slope is 1.0 is not an acceptable approach. Accuracy of physical property methods may be assessed through the analysis of standard reference materials, or alternatively, the suitability of the above approaches may be considered on a case-by-case basis.

precision

Definition— The precision of an analytical procedure is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of a homogeneous sample. The precision of an analytical procedure 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 of repeatability of the analytical procedure 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 (also known as ruggedness) 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.

Determination— The precision of an analytical procedure 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 using 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 by other supporting analytical procedures. [Note—Other reputable international authorities (IUPAC, AOAC-I) have preferred the term ―selectivity,‖ reserving ―specificity‖ for those procedures that are complet ely selective. ] For the tests discussed 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 impurities).

Assays: provide an exact result, which allows an accurate statement on the content or potency of the analyte in a sample.

Determination— In the case of qualitative analyses (identification tests), the ability to select between compounds of closely related structure that are likely to be present should be demonstrated. This 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 the case of analytical procedures 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.

In the case of the 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/base hydrolysis, and oxidation). In the case of the assay, the results should be compared; in the case of 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.

For validation of specificity for qualitative and quantitative determinations by spectroscopic methods, chapters related to topics such as near-infrared spectrophotometry, raman spectroscopy, and X-ray powder diffraction should be consulted.

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 procedures, 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 approach may be used as for noninstrumental procedures. In the case of procedures submitted for consideration as official compendial procedures, 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 procedures, 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 approach may be used as for noninstrumental procedures. In the case of procedures submitted for consideration as official compendial procedures, 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 and below the quantitation level. For example, if it is required that an analyte be assayed at the level of 0.1 mg per tablet, it should be demonstrated that the procedure will reliably quantitate the analyte 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 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 approach 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 procedure 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. Thus, in this section, ―linearity‖ refers to the linearity of the

relationship of concentration and assay measurement. In some cases, to attain linearity, the concentration and/or the measurement may be transformed. (Note that the weighting factors used in the regression analysis may change when a transformation is applied.) Possible transformations may include log, square root, or reciprocal, although other transformations are acceptable. If linearity is not attainable, a nonlinear model may be used. The goal is to have a model, whether linear or nonlinear, that describes closely the concentration-response relationship.

Definition of Range— The range of an analytical procedure is the interval between the upper and lower levels of analyte (including these levels) that have been demonstrated to be determined with a suitable level of precision, accuracy, and linearity using the procedure as written. The range is normally expressed in the same units as test results (e.g., percent, parts per million) obtained by the analytical procedure.

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). Data from the regression line itself may be helpful to provide 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 procedure is validated by verifying that the analytical procedure 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 acceptance criterion. 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 acceptance criteria for a controlled-release product cover a region from 30%, after 1 hour, and up to 90%, after 24 hours, the validated range would be 10% to 110% of the label claim).

The traditional definition of linearity, i.e., the establishment of a linear or mathematical relationship between sample concentration and response, is not applicable to particle size analysis. For particle size analysis, a concentration range is defined (instrument- and particle size-dependent) such that the measured particle size distribution is not affected by changes in concentration within the defined concentration range. Concentrations below the defined concentration range may introduce an error due to poor signal-to-noise ratio, and concentrations exceeding the defined concentration range may introduce an error due to multiple scattering.

robustness

Definition— The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in procedural parameters listed in the procedure documentation and provides an indication of its suitability during normal usage. Robustness may be determined during development of the analytical procedure.

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 procedure. One consequence of the evaluation of robustness and ruggedness should be that a series of system suitability parameters is

established to ensure that the validity of the analytical procedure is maintained whenever used. Typical variations are the stability of analytical solutions, different equipment, and different analysts. In the case of 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 procedure depend on the type of procedure being evaluated. They are especially important in the case of chromatographic procedures. 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 Validation

Compendial test requirements vary from highly exacting analytical determinations to subjective evaluation of attributes. Considering this broad variety, it is only logical that different test procedures require different validation schemes. This chapter covers only the most common categories of tests for which validation data should be required. These categories are as follows:

Category I— Analytical procedures for quantitation of major components of bulk drug substances or active ingredients (including preservatives) in finished pharmaceutical products.

Category II— Analytical procedures for determination of impurities in bulk drug substances or degradation compounds in finished pharmaceutical products. These procedures include quantitative assays and limit tests.

Category III — Analytical procedures for determination of performance characteristics (e.g., dissolution, drug release, etc.).

Category IV — Identification tests.

For each category, different analytical information is needed. Listed in Table 2 are data elements that are normally required for each of these categories.

Table 2. Data Elements Required for Validation

Analytical Performance Characteristics Category I Category II

Category III

Category IV Quantitative Limit Tests Accuracy

Yes Yes **No Precision

Yes Yes No Yes No Specificity

Yes Yes Yes *Yes Detection Limit

No No Yes *No Quantitation Limit

No Yes No *No Linearity

Yes Yes No *No Range Yes Yes **

No * May be required, depending on the nature of the specific test. Already established general procedures (e.g., titrimetric determination of water, bacterial endotoxins) should be verified to establish their suitability for use, such as their accuracy (and absence of possible interference) when used for a new product or raw material.

When validating physical property methods, consider the same performance characteristics required for any analytical procedure. Evaluate use of the performance characteristics on a case-by-case basis, with the goal of

determining that the procedure is suitable for its intended use. The specific acceptance criteria for each validation parameter should be consistent with the intended use of the method.

Physical methods may also be classified into the four validation categories. For example, validation of a quantitative spectroscopic method may involve

evaluation of Category I or Category II Analytical Performance Characteristics, depending on the method requirements. Qualitative physical property

measurements, such as particle size, surface area, bulk and tapped density,

which could impact performance characteristics, often best fit in Category III. Category IV Analytical Performance Characteristics usually applies to validation of qualitative identification spectroscopic methods. However, the various techniques may be used for different purposes, and the specific use of the method and characteristics of the material being analyzed should be considered when definitively applying a category to a particular type of method.

The validity of an analytical procedure can be verified only by laboratory studies. Therefore, documentation of the successful completion of such studies is a basic requirement for determining whether a procedure is suitable for its intended application(s). Current compendial procedures are also subject to regulations that require demonstration of suitability under actual conditions of use (see Verification of Compendial Procedures 1226for principles relative to the verification of compendial procedures). Appropriate documentation should accompany any proposal for new or revised compendial analytical procedures.

Auxiliary Information— Please check for your question in the FAQs before contacting USP.

Topic/Question Contact Expert Committee

General Chapter Horacio N. Pappa, Ph.D.

Principal Scientific

Liaison

1-301-816-8319 (GCPA2010) General Chapters - Physical Analysis

USP34–NF29 Page 778

Pharmacopeial Forum: Volume No. 35(2) Page 444

药物化学实验指导

磺胺嘧啶锌(Sulfadiazine-Zn) 与磺胺嘧啶银（Sulfadiazine-Ag ）的合成 一、目的要求： 了解拼合原理在药物结构修饰中的应用。 二、实验原理 磺胺嘧啶银为应用烧伤创面的磺胺药，对绿脓杆菌有强的抑制作用，其特点是保持了磺胺嘧啶与硝酸银二者的抗菌作用。除用于治疗烧伤创面感染和控制感染外，还可使创面干燥，结痂，促进愈合。但磺胺嘧啶银成本较高，且易氧化变质，故制成磺胺嘧啶锌，以代替磺胺嘧啶银。其化学名分别为2-（对氨基苯磺酰胺基）嘧啶银（SD-Ag ）、2-（对氨基苯磺酰胺基）嘧啶锌（SD-Zn ），化学结构式分别为： NH 2 SO 2N N N NH 2 SO 2N N N NH 2SO 2N Zn N N 磺胺嘧啶银为白色或类白色结晶性粉末，遇光或遇热易变质。 在水、乙醇、氯仿或乙醚中均不溶。磺胺嘧啶锌为白色或类白色粉末，在水、乙醇、氯仿、或乙醚中均不溶。 合成路线如下： NH 3.H 2O H 2O ++SO 2N N N NH 2 SO 2N N N NH 4 NH 2 SO 2N N N NH 4 NH 2 NH 2SO 2N N N NH 2 SO 2N N N NH 2SO 2N Zn N N

三、实验方法 （一）磺胺嘧啶银的制备 取磺胺嘧啶5 g，置50 mL烧杯中，加入10% 氨水20 mL溶解。再称取AgNO33.4 g置50 mL烧杯中，加10 mL氨水溶解，搅拌下，将AgNO3-氨水溶液倾入磺胺嘧啶-氨水溶液中，片刻析出白色沉淀，抽滤，用蒸馏水洗至无Ag+反应，得本品。干燥，计算收率。 （二）磺胺嘧啶锌的制备 取磺胺嘧啶5 g，置100 mL烧杯中，加入稀氨水（4 mL浓氨水加入25 mL水），如有不溶的磺胺嘧啶，再补加少量浓氨水（约1 mL左右）使磺胺嘧啶全溶。另称取硫酸锌3 g，溶于25 mL水中，在搅拌下倾入上述磺胺嘧啶氨水溶液中，搅拌片刻析出沉淀，继续搅拌5 min ，过滤，用蒸馏水洗至无硫酸根离子反应（用0.1 M氯化钡溶液检查），干燥，称重，计算收率。 （三）结构确证 1. 红外吸收光谱法、标准物TLC对照法。 2. 核磁共振光谱法。 注释： 合成磺胺嘧啶银时，所有仪器均需用蒸馏水洗净。 思考题： 1. SD-Ag及SD-Zn的合成为什么都要先作成铵盐？ 2. 比较SD-Ag及SD-Zn的合成及临床应用方面的优缺点。

美国FDA分析方法验证指南中英文对照

I. INTRODUCTION This guidance provides recommendations to applicants on submitting analytical procedures, validation data, and samples to support the documentation of the identity, strength, quality, purity, and potency of drug substances and drug products. 1. 绪论 本指南旨在为申请者提供建议，以帮助其提交分析方法，方法验证资料和样品用于支持原料药和制剂的认定，剂量，质量，纯度和效力方面的文件。 This guidance is intended to assist applicants in assembling information, submitting samples, and presenting data to support analytical methodologies. The recommendations apply to drug substances and drug products covered in new drug applications (NDAs), abbreviated new drug applications (ANDAs), biologics license applications (BLAs), product license applications (PLAs), and supplements to these applications. 本指南旨在帮助申请者收集资料，递交样品并资料以支持分析方法。这些建议适用于NDA，ANDA，BLA，PLA及其它们的补充中所涉及的原料药和制剂。 The principles also apply to drug substances and drug products covered in Type II drug master files (DMFs). If a different approach is chosen, the applicant is encouraged to discuss the matter in advance with the center with product jurisdiction to prevent the expenditure of resources on preparing a submission that may later be determined to be unacceptable. 这些原则同样适用于二类DMF所涉及的原料药和制剂。如果使用了其它方法，鼓励申请者事先和FDA药品评审中心的官员进行讨论，以免出现这种情况，那就是花了人力物力所准备起来的递交资料后来发现是不可用的。 The principles of methods validation described in this guidance apply to all types of analytical procedures. However, the specific recommendations in this guidance may not be applicable to certain unique analytical procedures for products such as biological, biotechnological, botanical, or radiopharmaceutical drugs. 本指南中所述的分析方法验证的原则适用于各种类型的分析方法。但是，本指南中特定的建议可能不适用于有些产品所用的特殊分析方法，如生物药，生物技术药，植物药或放射性药物等。 For example, many bioassays are based on animal challenge models, 39 immunogenicity assessments, or other immunoassays that have unique features that should be considered when submitting analytical procedure and methods validation information. 比如说，许多生物分析是建立在动物挑战模式，免疫原性评估或其它有着独特特性的免疫分析基础上的，在递交分析方法和分析方法验证资料时需考虑这些独特的性质。Furthermore, specific recommendations for biological and immunochemical tests that may be necessary for characterization and quality control of many drug substances and drug products are beyond the scope of this guidance document. 而且，许多原料药和制剂的界定和质量控制所需的生物和免疫化学检测并不在本指南的范围之内。 Although this guidance does not specifically address the submission of analytical procedures and validation data for raw materials, intermediates, excipients, container closure components, and other materials used in the production of drug

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于1996 年11 月颁了《分析方法的验证：方法学》 (Validation of Analytical Procedures．Methodology)；美国药典第24 版 <1225>规定了《药典方法的验证)) (Validation of Compendial Methods)；中国药典 2000 版附录ⅩⅨA 规定了《药品质量标准分析方法验证》。 这些法规、规定从不同方面对检验方法的验证作了规定，它们是实施检验方法验证的重要依 据。本章将着重讨论检验方法验证的基本内容，并以示例形式介绍验证的基本实践，以使本 章的内容具有可操作性。 方法验证有以下几个先决条件，在进行方法验证以前，必须逐条进行检查［1］。 (1) 仪器已经过校正且在有效期内。 (2) 人员人员应经过充分的培训，熟悉方法及所使用的仪器。 (3) 参照品参照品的来源一般有 3 个：购自法定机构(如中国生物制品检定所)的法定参 照品；购自可靠的供应商，如Sigma，Merck 等；自备参照品，其纯度和性能可自行检测或 由法定检验机构检测。 (4) 材料所用材料，包括试剂、实验用容器等，均应符合试验要求，不给实验带来污 染、误差。如进行高效液相色谱分析时，所用试剂应为色谱级；检查铁盐时使用的盐酸不得 含有铁盐等。 (5) 稳定性应在开始进行方法验证前考察试验溶液和试剂的稳定性，确保在检验周期 内试验溶液和试剂是稳定的。使用自动进样器，一般是预先配制好一系列样品溶液置进样器 中，依次进样。这时要确保进样周期内样品溶液是稳定的。

中药化学实验指导—常用实验操作技术

常用实验操作技术 （一）浸渍法操作 冷浸法取药材粗粉，置适宜容器中，加入一定量的溶剂如水、酸水、碱水或稀醇等，密闭，时时搅拌或振摇，在室温条件下浸渍1～2天或规定时间，使有效成分浸出，滤过。药材再加入适量溶剂浸泡2～3次，使有效成分大部分浸出。然后将药渣充分压榨、滤过，合并滤液，经浓缩后可得提取物。 温浸法具体操作与冷浸法基本相同，但温浸法的浸渍温度一般在40～60℃之间，浸渍时间短，却能浸出较多的有效成分。由于温度较高，浸出液冷却后放置贮存常析出沉淀，为保证质量，需滤去沉淀后再浓缩。 （二）渗漉法操作 1．渗漉装置常用的渗漉装置（实图-2），渗漉筒一般为圆柱形或圆锥形，筒的长度为筒直径的2～4倍。渗漉提取膨胀性不大的药材时用圆柱形渗漉筒；圆锥形渗漉筒则用于膨胀性大的药材渗漉提取。 2．操作方法将药材粗粉放在有盖容器内，再加入药材粗粉量60％～70％的浸出溶剂均匀湿润后，密闭，放置15分钟至数小时，使药材充分膨胀后备用。另取脱脂棉一团，用浸出液润湿后，铺垫在渗漉筒的底部，然后将已湿润膨胀的药材粗粉分次装入渗漉筒中，每次装药后，均须摊匀压平。松紧程度视药材质地及浸出溶剂而定，若为含水量较多的溶剂宜压松些，含醇量高的溶剂则可压紧些。药粉装完后，用滤纸或纱布将药材面覆盖，并加一些玻璃珠或碎瓷片等重物，以防加入溶剂时药粉被冲浮起来。然后向渗漉筒中缓缓加入溶剂，并注意应先打开渗漉筒下方浸液出口之活塞，以排除筒内空气，待溶液自下口流出时，关闭活塞。流出的溶剂应再倒回筒内，并继续添加溶剂至高出药粉表面数厘米，加盖放置24～48小时，使溶剂充分渗透扩散。开始渗漉时，漉液流出速度如以1000g药粉计算，每分钟流出1～3ml或3～5ml为宜。渗漉过程中需随时补充新溶剂，使药材中有效成分充分浸出。渗漉溶剂的用量一般为1∶4～8（药材粉末∶渗漉溶剂）。 实图-2渗漉装置

FDA分析方法验证指南

（中英对照）美国FDA 分析方法验证指南

目 录 1.绪论 (4) II.背景 (5) III.分析方法的类型 (7) A. 法定分析方法 (7) B. 替代分析方法 (7) C. 稳定性指示分析 (8) IV 标准品 (8) A．标准品的类型 (8) B．分析报告单 (9) C．标准品的界定 (9) V．IND中的分析方法验证 (11) VI．NDA，ANDA，BLA和PLA中分析方法的内容和格式 (12) A．基本方法 (12) B．取样 (12) C．仪器和仪器参数 (12) D．试剂 (13) E．系统适应性实验 (13) F．标准品的制备 (14) H．操作过程 (14) J．计算 (14) K.结果报告 (14) VII.NDA，ANDA，BLA和PLA中的分析方法验证 (16) A．非药典分析方法 (16) 1)验证项目 (16) 2)其它分析方法验证信息 (17) a.耐用性 (18) b.强降解实验 (19) c.仪器输出/原始资料 (19)

3)各类检测的推荐验证项目 (21) B.药典分析方法 (24) VIII.统计分析 (25) A．基本原则 (25) B.对比研究 (25) C.统计 (25) IX．再验证 (26) X．分析方法验证资料：内容和数据处理 (27) A．分析方法验证资料 (27) B.样品的选择和运输 (29) C.各方职责 (30) XI．方法学 (32) A.高效液相色谱(HPLC) (32) B.气相色谱(GC) (35) C.分光光度法，光谱法和相关的物理方法 (37) D.毛细管电泳(CE) (37) E.旋光度 (39) F.粒径分析相关的分析方法 (40) G.溶出度 (41) H.其它仪器分析方法 (43) 附录A NDA, ANDA, BLA 和PLA申请的内容 (44) 附录B 析方法验证的问题和延误 (45) 参考文献 (46) 术语表 (48)

分析方法验证

分析方法验证 一．方法验证概述 1方法验证概念解析 2各方指导原则 3方法验证的常见困难 4风险管理在分析方法中的应用 5限度与定量验证的区别与适用情况 6方法验证的前期准备 7方法验证的方案和报告 首先申明是方法验证：ICHQ2，分析方法的验证是为了证明该方法与预期的目的相一致。中国药典，药品质量标准分析方法验证的目的是证明采用的方法适合于相应的检测要求。USP1225，通过实验室研究确认方法的性能参数可以满足预期的检测要求。 什么是检测要求：结实（耐用性好）、准确、稳定 是否需要验证？ 需要经过验证的：含量测定、有关物质、残留溶剂、基因毒杂质、其他特定杂质、鉴别（仪器）、溶出度、释放度、粒径（激光衍射法）、其他采用液相色谱及光谱法等检测项目。 不需要经过验证的：炽灼残渣、干燥失重、鉴别（化学法）、性状、水分、酸碱度、PH、其他理化检测。

注意事项：方法是否需要验证，应当基于风险评估的判断，惯例只是对于风险的一致认知，并不适用于所有情况。 注册用方法验证的一般要求 代表性的样品：验证用到的样品应当能够代表上市批的质量，工艺一致，批量相当。 规范的过程：验证过程应当符合GMP或GMP 类似要求方法确认与转移相关指导原则 12015年版中国药典四部9101 2.化学药物质量控制分析方法验证技术指导原则 https://www.sodocs.net/doc/b23007719.html,P1225 4.ICHQ2 5.FDA的Analytical procedures and method validation 方法验证的常见困难： 1 已经验证的方法转移失败。 2 已经验证的方法在日常检测过程中不适用。 3方法验证过程中发现方法不适用 4 某些验证项目需要多次验证才能获得满意的结果。 分析方法风险应对前置于方法开发阶段 1在方法设计阶段应当预测全生命周期内可能存在的风险，如方法转移、可能的OOT/OOS、系统适用性试验失败原因、监管法规的变化、技术进步等。 2 收集相关方的需求，包括QC实验室、合成部门、制