ICH指导原则之方法论

INTRODUCTION 简介

This document is complementary to the parent document, which presents a discussion of the characteristics that should be considered during the validation of analytical procedures. Its purpose is to provide some guidance and recommendations on how to consider the various validation characteristics for each analytical procedure.In some cases (for example, demonstration of specificity), the overall capabilities of a number of analytical procedures in combination may be investigated in order to ensure the quality of the drug substance or drug product. In addition, the document provides an indication of the data, which should be presented in a registration application.

本文作为前文的补充，旨在讨论在分析方法验证过程中在每一个具体的项目需要考虑哪些内容。本文的目的是就不同类型的验证该涵盖哪些项目提供一个指导原则和建议。以专属性为例，为了确保原料药或制剂的质量，需要考察分析过程对化合物中杂质的全面综合分析能力。另外，文件提供的数据应该包含在注册申请材料中。

All relevant data collected during validation and formulae used for calculating validation characteristics should be submitted and discussed as appropriate.

验证报告中的所有数据及每个验证项目的计算公式应一同提交并进行适当的论述(即得出相应结论)。

Approaches other than those set forth in this guideline may be applicable and acceptable. It is the responsibility of the applicant to choose the validation procedure and protocol most suitable for their product. However it is important to remember that the main objective of validation of an analytical procedure is to demonstrate that the procedure is suitable for its intended purpose. Due to their complex nature, analytical procedures for biological and biotechnological products in some cases may be approached differently than in this document.

本文阐述的原则之外的原则应该适用并可接受。申请者的职责是制定最适合申报产品的验证项目及验证方案。最重要的是记住分析方法验证的主要目的是证明该分析程序能达到预期目标。由于生物制品和生物科技产品本身的复杂性，有时候，其分析方法的验证也可能与本文

Well-characterized reference materials, with documented purity, should be used throughout the validation study. The degree of purity necessary depends on the intended use.

验证研究用到的参比物质需要经过完全鉴定并标定纯度。所需的纯度取决于其应用类型(预期用途)。

In accordance with the parent document, and for the sake of clarity, this document considers the various validation characteristics in distinct sections. The arrangement of these sections reflects the process by which an analytical procedure may be developed and evaluated.

与前文相同，该文件考虑了各个独立章节的不同验证项目。这些章节的安排也反映了分析方法的建立和评估的过程。

In practice, it is usually possible to design the experimental work such that the appropriate validation characteristics can be considered simultaneously to provide a sound, overall knowledge of the capabilities of the analytical procedure, for instance: specificity, linearity, range, accuracy and precision.

事实上，设计试验的时候，一些适当的验证项目可以同时考虑，以便对分析方法的能力提供合理的，全面的了解(依据)，例如：专属性、线性、范围、准确度、精密度。

provide a……knowledge of 为……提供依据

1. SPECIFICITY 专属性

An investigation of specificity should be conducted during the validation of identification tests, the determination of impurities and the assay. The procedures used to demonstrate specificity will depend on the intended objective of the analytical procedure.

鉴别测试、杂质和含量测试方法的验证都应考察方法的专属性。证明专属性的方法取决于分析方法的预期目的。

It is not always possible to demonstrate that an analytical procedure is specific for a

analytical procedures is recommended to achieve the necessary level of discrimination.

一般来说，某一种分析方法不太可能完全证明其对某一特定被分析物具有专属性。这种情况下，建议采用两种或两种以上的分析方法以确保完全鉴别水平。

1.1. Identification 鉴别

Suitable identification tests should be able to discriminate between compounds of closely related structures, which are likely to be present. The discrimination of a procedure may be confirmed by obtaining positive results (perhaps by comparison with a known reference material) from samples containing the analyte, coupled with negative results from samples, which do not contain the analyte. In addition, the identification test may be applied to materials structurally similar to or closely related to the analyte to confirm that a positive response is not obtained. The choice of such potentially interfering materials should be based on sound scientific judgment with a consideration of the interferences that could occur.

合适的鉴别方法应该能够区分可能存在的结构相近的化合物。可以同已知参考物质进行比较，从含有被分析物的样品得到的正的结果和不含被分析物的样品得到的负的结果来确定。此外，鉴别测试也可以用结构相近或相关的物质测试得不到正的反应来证实。在考虑可能会造城干扰的前提下，应根据合理科学的判断来选择可能存在的干扰物。

likely to be present：可能存在的；discrimination：比较；

1.2. Assay and Impurity Test(s) 含量和杂质测定

For chromatographic procedures, representative chromatograms should be used to demonstrate specificity and individual components should be appropriately labelled. Similar considerations should be given to other separation techniques.

在色谱法测定中，需要用有代表性的图谱证明专属性，并恰当的注明每一个成分。其它的分离技术也应如此。

Critical separations in chromatography should be investigated at an appropriate level. For critical separations, specificity can be demonstrated by the resolution of the two

色谱分离法应在一定程度上考察关键性的分离。对关键性的分离，可用两个洗脱程度最接近的化合物的分离度来证明其专属性。

In cases where a non-specific assay is used, other supporting analytical procedures should be used to demonstrate overall specificity. For example, where a titration is adopted to assay the drug substance for release, the combination of the assay and a suitable test for impurities can be used.

当采用非专属性的方法测定含量时，应采用辅助性分析方法来证明整个方法具有专属性。例如用滴定法测定放行原料药的含量，可结合使用合适的杂质检测方法。

supporting analytical procedures：辅助性分析方法；overall specificity：整体专属性；combination：结合，联合；

The approach is similar for both assay and impurity tests:

下述方法均适用于含量和杂质检测。

is similar for：适用于

1.2.1 Impurities are available 可以得到杂质的情况

For the assay, this should involve demonstration of the discrimination of the analyte in the presence of impurities and/or excipients; practically, this can be done by spiking pure substances (drug substance or drug product) with appropriate levels of impurities and/or excipients and demonstrating that the assay result is unaffected by the presence of these materials (by comparison with the assay result obtained on unspiked samples).

对含量检测，专属性应该包括提供被分析物在杂质和/或赋形剂存在时能被区分的证明；实际操作时，可通过向纯物质(原料药或制剂)中加入一定量的杂质和/或赋形剂的检测结果和未添加杂质和/或赋形剂的纯物质的检测结果进行对比以此证明这些杂质和/或赋形剂的存在不会对含量检测结果造成影响。

discrimination：区分

For the impurity test, the discrimination may be established by spiking drug substance or

these impurities individually and/or from other components in the sample matrix.

对杂质检测，可以向原料药或制剂中加入一定量的杂质，证明各杂质能够分离且能与样品中的其它组分分离。

1.2.2 Impurities are not available 无法得到杂质的情况

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.: pharmacopoeial method or other validated analytical procedure (independent procedure). As appropriate, this should include samples stored under relevant stress conditions: light, heat, humidity, acid/base hydrolysis and oxidation.

如果无法得到杂质或降解产物的对照品，检测方法的专属性可以通过将含有一定量的杂质或降解产物的样品的检测结果与另一种成熟的检测方法——如药典方法或经验证的其它方法(独立的方法)——的检测结果进行比较来证明。必要时，应该包括放置在强降解试验条件，即强光，高温，高湿，酸/碱水解及氧化条件下的样品测试。

As appropriate：必要时；

-For the assay, the two results should be compared;

-对含量检测，需要对比两种方法的检测结果

-For the impurity tests, the impurity profiles should be compared.

-对杂质检测，需要对比杂质概况

Peak purity tests may be useful to show that the analyte chromatographic peak is not attributable to more than one component (e.g., diode array, mass spectrometry).

峰纯度测试是非常有用的，它能显示被测物的色谱峰是一个成分还是多个成分(如二极管阵列，质谱)。

2. LINEARITY 线性

A linear relationship should be evaluated across the range (see section 3) of the analytical procedure. It may be demonstrated directly on the drug substance (by dilution of a standard stock solution) and/or separate weighings of synthetic mixtures of the drug product components, using the proposed procedure. The latter aspect can be studied during investigation of the range.

检测方法的线性关系应该在范围内(见章节3)进行评价。线性研究可通过所建议的分析方法，直接对原料药(用标准储备液稀释)和/或分别称取制剂组分的混合物测试来进行。后者应在方法的范围内进行研究。

Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. If there is a linear relationship, test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares. In some cases, to obtain linearity between assays and sample concentrations, the test data may need to be subjected to a mathematical transformation prior to the regression analysis. Data from the regression line itself may be helpful to provide mathematical estimates of the degree of linearity.

线性应关系应以信号对被测物浓度或含量作图，根据图形是否呈线性来评估。如果呈线性关系，测试结果应用适当的统计学方法进行评估，例如用最小二乘法进行线性回归计算。在某些情况下，为了使含量与样品浓度呈线性关系，在回归分析前需要对测试数据进行数学转化。由线性回归评估所得的数据本身又有助于精确的评价线性的程度。

In some cases：在某些情况下，有时候；

The correlation coefficient, y-intercept, slope of the regression line and residual sum of squares should be submitted. A plot of the data should be included. In addition, an analysis of the deviation of the actual data points from the regression line may also be helpful for evaluating linearity.

相关系数，y轴上的截距，回归曲线的斜率以及剩余方差应包含在递交材料里。还应包括数据图表。另外，实际数据点与回归曲线的偏差也有助于对线性进行评价。

Some analytical procedures, such as immunoassays, do not demonstrate linearity after any transformation. In this case, the analytical response should be described by an appropriate function of the concentration (amount) of an analyte in a sample.

一些分析方法，如免疫测定法，在任何转换后，均不能证明呈线性。在这种情况下，分析的相应值应用被分析物的浓度(数量)的适当函数来表示。

For the establishment of linearity, a minimum of 5 concentrations is recommended. Other approaches should be justified.

为建立线性，建议至少用5个浓度。若用其他方法应证明其合理性。

3. RANGE 范围

The specified range is normally derived from linearity studies and depends on the intended application of the procedure. It is established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy and precision when applied to samples containing amounts of analyte within or at the extremes of the specified range of the analytical procedure.

特定的范围一般是从线性研究中得到的，它依赖于分析方法的应用目的。确定范围的方法是：样品中含有被分析物的量在分析方法规定的范围内或在范围末端，该分析方法均能获得良好的线性，精密度及准确度。

The following minimum specified ranges should be considered:

以下是应考虑的最小规定范围：

-for the assay of a drug substance or a finished (drug) product: normally from 80 to 120 percent of the test concentration;

-对原料药或成品药(制剂)的含量测定：一般应在测试浓度的80%～120%；

-for content uniformity, covering a minimum of 70 to 130 percent of the test concentration, unless a wider more appropriate range, based on the nature of the dosage form (e.g., metered dose inhalers), is justified;

主要是根据剂型的特点(如定量吸入剂)；

-for dissolution testing: +/-20 % over the specified range;

-对溶出度测试，应为规定范围的+/-20%；

e.g., if the specifications for a controlled released product cover a region from 20%, after 1 hour, up to 90%, after 24 hours, the validated range would be 0-110% of the label claim.

例如：如果是控释剂，规定1小时后达到20%，24小时后达到90%，它的验证范围应为标示量的0～110%。

-for the determination of an impurity: from the reporting level of an impurity1 to 120% of the specification;

-对杂质测定，应为杂质的报告水平至标准规定的120%；

-for impurities known to be unusually potent or to produce toxic or unexpected pharmacological effects, the detection/quantitation limit should be commensurate with the level at which the impurities must be controlled;

-对已知有异常功效的，有毒的或者有意外药理作用的杂质，其检测限度和定量限度应与该杂质必须被控制的水平相当。

Note: for validation of impurity test procedures carried out during development, it may be necessary to consider the range around a suggested (probable) limit.

注意：在研制阶段进行杂质检测方法验证时，有必要根据建议(可能)的限度水平来考虑范围；

-if assay and purity are performed together as one test and only a 100% standard is used, linearity should cover the range from the reporting level of the impurities1 to 120% of the assay specification.

1see chapters “Reporting Impurity Content of Batches” of the corresponding ICH-Guidelines: “Impurities in New Drug Substances” and “Impurities in New Drug

-如果一个试验同时进行含量和纯度检测，且仅使用100%的标准品，线性范围应覆盖杂质的报告水平(见相应ICH指南“新原料药中的杂质”和“新制剂中的杂质”中“批杂质含量的报告”章节)至含量指标120%。

4. ACCURACY 准确度

Accuracy should be established across the specified range of the analytical procedure.

应在分析方法规定的范围内建立(考察方法的)准确度。

4.1. Assay 含量

4.1.1 Drug Substance 原料药

Several methods of determining accuracy are available:

以下几种方法可用于测定准确度：

a) application of an analytical procedure to an analyte of known purity (e.g. reference

material);

用该分析方法测定已知纯度的被分析物(例如参照物质)；

b) comparison of the results of the proposed analytical procedure with those of a second

well-characterized procedure, the accuracy of which is stated and/or defined (independent procedure, see 1.2.);

用建议采用的分析方法的结果与另一种完全验证过的方法的结果作对比，对比的方法的准确度是规定的(一定的)和/或已定义的(独立的方法，见1.2节)

c) accuracy may be inferred once precision, linearity and specificity have been

established.

准确度可以在精密度，线性及专属性建立之后推论得到；

4.1.2 Drug Product 制剂

Several methods for determining accuracy are available:

以下几种方法可用于测定准确度：

a) application of the analytical procedure to synthetic mixtures of the drug product

added;

用该分析方法测定按处方量制成的混合物，其中加入了已知量的待测原料药。

b) in cases where it is impossible to obtain samples of all drug product components , it may

be acceptable either to add known quantities of the analyte to the drug product or to compare the results obtained from a second, well characterized procedure, the accuracy of which is stated and/or defined (independent procedure, see 1.2.);

如果不能得到制剂的所有成分，向制剂中加入已知量的被测物或者与另一种经过完整验证过的准确度是规定的(一定的)和/或已定义的方法的结果作对比，(独立的方法，见1.2节)也是可以接受的；

c) accuracy may be inferred once precision, linearity and specificity have been

established.

准确度可以在精密度，线性及专属性建立之后推论得到；

4.2. Impurities (Quantitation) 杂质(定量)

Accuracy should be assessed on samples (drug substance/drug product) spiked with known amounts of impurities.

准确度可以通过向样品(原料药/制剂)中加入已知量杂质的方法来评价。

In cases where it is impossible to obtain samples of certain impurities and/or degradation products, it is considered acceptable to compare results obtained by an independent procedure (see 1.2.). The response factor of the drug substance can be used.

如果无法得到杂质和或降解产物的样品，可以通过与其它独立方法(见1.2节)的检测结果进行对比评估其准确度。可以使用原料药的响应因子。

It should be clear how the individual or total impurities are to be determined e.g., weight/weight or area percent, in all cases with respect to the major analyte.

需要说明单杂和总杂是如何测定的，如相对于主要被分析物所占的质量分数或面积百分比。

4.3. Recommended Data 可接受数据(数据要求)

Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3

of the total analytical procedure).

准确度的评价需要在方法的线性范围内的三种浓度至少测定九次(按完整分析步骤对三种浓度每种浓度重复进样三次)。

Accuracy should be reported as percent recovery by the assay of known added amount of analyte in the sample or as the difference between the mean and the accepted true value together with the confidence intervals.

准确度应以向样品中加入已知量的被测物所得的百分回收率或者平均值和可接受真实值之间的差值及置信区间来报告。

5. PRECISION 精密度

Validation of tests for assay and for quantitative determination of impurities includes an investigation of precision.

含量和杂质的定量分析需要考察方法的精密度。

5.1. Repeatability 重复性

Repeatability should be assessed using:

重复性可以通过一下方法进行考察：

a) a minimum of 9 determinations covering the specified range for the procedure (e.g., 3

concentrations/3 replicates each);

在方法的线性范围内至少检测九次(三种浓度每种浓度重复进样三次)；

b) or a minimum of 6 determinations at 100% of the test concentration.

以100%测试浓度至少检测六次。

5.2. Intermediate Precision 中间精密度

The extent (程度) to which intermediate precision should be established depends on the circumstances under which the procedure is intended to be used. The applicant should establish the effects of random events on the precision of the analytical procedure. Typical variations to be studied include days, analysts, equipment, etc. It is not considered necessary to study these effects individually. The use of an experimental design (matrix) is encouraged.

中间精密度的考察程度应根据分析方法的操作环境而定。申请者应确定(弄清楚)随机时间对分析方法的精密度的影响。需要研究的典型变化有：日期，分析者，仪器等。没有必要逐项

5.3. Reproducibility 重现性

Reproducibility is assessed by means of (通过) an inter-laboratory trial. Reproducibility should be considered in case of the standardization of an analytical procedure, for instance, for inclusion of procedures in pharmacopoeias. These data are not part of the marketing authorization dossier.

重现性可通过实验室之间的试验进行评估。如果方法需要标准化，例如药典方法，则应考虑重现性。这些资料不是上市申请文档的一部分(申报注册不需要考察药典收录方法的重现性，这是药典委需要考虑的问题)。

5.4. Recommended Data 数据要求

The standard deviation, relative standard deviation (coefficient of variation) and confidence interval should be reported for each type of precision investigated.

每一种精密度研究中都应报告标准偏差，相对标准偏差(变异系数)和置信区间。

6. DETECTION LIMIT 检测限

Several approaches for determining the detection limit are possible, depending on whether the procedure is a non-instrumental or instrumental. Approaches other than those listed below may be acceptable.

根据检测方法是用仪器分析还是非仪器分析，可用几种方法来确定检测限。除了下面所列的方法外，其它的分析方法也可能被接收。

6.1. Based on Visual Evaluation 视觉判定(根据直观评价)

Visual evaluation may be used for non-instrumental methods but may also be used with instrumental methods.

视觉判定可用于非仪器分析方法，也可用于仪器分析方法。

The detection limit is determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected.

检测限的确定是通过一系列已知浓度的分析物样品进行分析，并以能准确测得被分析物的最小水平来建立。

6.2. Based on Signal-to-Noise

Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit.

6.3 Based on the Standard Deviation of the Response and the Slope

The detection limit (DL) may be expressed as:

3.3 σ

DL =

S

where σ = the standard deviation of the response

S = the slope of the calibration curve

The slope S may be estimated from the calibration curve of the analyte. The estimate of s may be carried out in a variety of ways, for example:

6.3.1 Based on the Standard Deviation of the Blank

Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the standard deviation of these responses.

6.3.2 Based on the Calibration Curve

A specific calibration curve should be studied using samples containing an analyte in the range of DL. The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation.

The detection limit and the method used for determining the detection limit should be presented. If DL is determined based on visual evaluation or based on signal to noise ratio, the presentation of the relevant chromatograms is considered acceptable for justification.

In cases where an estimated value for the detection limit is obtained by calculation or extrapolation, this estimate may subsequently be validated by the independent analysis of a suitable number of samples known to be near or prepared at the detection limit.

7. QUANTITATION LIMIT

Several approaches for determining the quantitation limit are possible, depending on whether the procedure is a non-instrumental or instrumental. Approaches other than those listed below may be acceptable.

7.1. Based on Visual Evaluation

Visual evaluation may be used for non-instrumental methods but may also be used with instrumental 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 quantified with acceptable accuracy and precision.

7.2. Based on Signal-to-Noise Approach

This approach can only be applied to analytical procedures that exhibit baseline noise.

Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and by establishing the minimum concentration at which the analyte can be reliably quantified. A typical signal-to-noise ratio is 10:1.

The quantitation limit (QL) may be expressed as:

10 σ

QL =

S

where σ = the standard deviation of the response

S = the slope of the calibration curve

The slope S may be estimated from the calibration curve of the analyte. The estimate of s may be carried out in a variety of ways for example:

7.3.1 Based on Standard Deviation of the Blank

Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the standard deviation of these responses.

7.3.2 Based on the Calibration Curve

A specific calibration curve should be studied using samples, containing an analyte in the range of QL. The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation.

7.4 Recommended Data

The quantitation limit and the method used for determining the quantitation limit should be presented.

The limit should be subsequently validated by the analysis of a suitable number of samples known to be near or prepared at the quantitation limit.

8. ROBUSTNESS

depends on the type of procedure under study. It should show the reliability of an analysis with respect to deliberate variations in method parameters.

If measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure. One consequence of the evaluation of robustness should be that a series of system suitability parameters (e.g., resolution test) is established to ensure that the validity of the analytical procedure is maintained whenever used.

Examples of typical variations are:

典型变化的例子：

-stability of analytical solutions;

-分析溶液的稳定性

-extraction time.

-提取时间

In the case of liquid chromatography, examples of typical variations are:

在液相色谱条件下的典型变化的例子：

-influence of variations of pH in a mobile phase;

-流动相pH值变化的影响

-influence of variations in mobile phase composition;

-不同流动相配比的影响；

-different columns (different lots and/or suppliers);

-不同的柱子(不同的批号/供应商)

-temperature;

-温度

-flow rate.

-流速

In the case of gas-chromatography, examples of typical variations are:

在气相色普条件下的典型变化的例子：

-不同的柱子(不同的批号/供应商)

-temperature;

-柱温

-flow rate.

-流速

9. SYSTEM SUITABILITY TESTING 系统适应性

System suitability testing is an integral part of many analytical procedures. The 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 validated. See Pharmacopoeias for additional information.

对于很多分析方法来说，系统适应性是主要组成部分。该项测试是基于仪器，电子设备，分析操作以及被测样品组成一个完整系统的概念并以此系统作为评价的基准。系统适应性试验参数根据被验证的方法类型中一个特定的规程来确定。具体的信息可以参考药典。