Analytical Method Validation

您传达的分析结果可能会带来深远的后果，除了涉及法律事务的决策外，还可以为使用商业产品，食品和自然资源的使用安全而做出决定。

作为负责任的分析师，在您在实验室进行分析之前，您应确保您的结果是真实的并且可以接受。只有在适当验证目的的方法时，才能实现此关键目标。

分析方法验证establishes documented evidence that the procedure adopted for a test is fit for the intended purpose in terms of quality, reliability and consistency of results.

什么是分析方法验证？

分析方法验证establishes documented evidence that the procedure adopted for a test is fit for the intended purpose in terms of quality, reliability and consistency of results.

换句话说，如果在世界各地的任何其他实验室中都采用了相同的方法，在相同的条件和控制参数下，结果应一致。

每种方法都需要验证：

• Before putting to routine use
• 当更改分析条件（例如技术变化）时，所需浓度范围或样品矩阵的变化
• Whenever changes are made to an existing procedure

It is important to collect relevant information on analysis requirements before you plan validation of the procedure

• Components to be detected
• Expected concentration levels
• 所需的检测和定量水平
• 样品矩阵的性质
• 使用的分析技术类型
• 所需程度的精度和准确性

方法验证的参数

在进行之前方法验证过程, it should be ascertained using reference standards that the response of the analytical measurement is on account of the analyte of interest and not due to any other chemical species.

After this has been established, you may proceed with the validation which evaluates the following method-specific features. Here are the various方法验证参数.

• 选择性和特异性
• 线性
• Range
• Accuracy
• 精确
• Limit of quantification
• 坚固
• 坚固

We shall discuss each of the分析方法验证参数详细,这样您就可以更好地理解分析化学ical method validation.

Selectivity and Specificity

选择性和特异性通常是代名词的，但这些术语不同。特异性是该技术对样品中存在的特定分析物的明确响应，而选择性可以适用于对具有相似化学和物理特征的一组分析物的集体响应。

The method selected should meet the criteria for selectivity and sensitivity in presence of deliberately added interferents that can be expected in the sample under investigation.

Selectivity is more frequently used as analytical techniques and is seldom specific to only one analyte. Further, selectivity should not be affected by interfering species such as degradation products, impurities and other matrix components.

线性

线性性是指分析程序产生与所需浓度水平内样品中分析物浓度范围直接成比例的结果的能力。

• 线性should be determined using a minimum of 6 standards whose concentration spans from 80% to 120% of expected concentration level.
• 线性report should include slope of line, linear range and correlation coefficient data. Correlation coefficient r should be greater than or equal to 0.99 in the working range

Range

Operating range is deduced from the calibration plot. It is the interval between the upper and lower concentration of analyte falling in the linear range. The results corresponding to this range demonstrate acceptable levels of precision, accuracy and linearity.

Accuracy

• Degree to which the determine value of analyte corresponds to the true value.
• 准确性在预期浓度范围内会有所不同。
• It should be determined using a working or reference standards in the 80% – 120% level of expected range
• 精度由：

1. 分析已知浓度样本并与真实值进行比较
2. Spiking a blank (Sample having all components except the analyte) and comparing with the expected result.
3. 确定样品浓度的标准加法方法。添加了已知量的分析物，并再次确定浓度。将两个浓度值的差异与添加分析物的实际值进行比较。

• 还可以通过将测试结果与使用其他经过验证的测试程序获得的测试结果进行比较来定义精度

精确

• 精确expresses closeness of a series of measurements of the same sample under identical conditions
• 高度的精度不一定意味着高度准确性
• 精确is expressed as variance, standard deviation or as coefficient of variation of a series of measurements
• Minimum of five replicate sample determinations should be carried out

检测极限

• 可以检测但不一定量化的分析物的最低量
• Lowest concentration of calibration standard which produces a peak response corresponding to the analyte should be measured at least 6 to 10 times. Average response (X) and standard deviation (SD) are required to calculate limit of detection

检测极限= x +（3SD）

• 检测极限时的信号与噪声比至少应为3：1

Limit of Quantitation

• Lowest amount of the analyte that can be quantitatively determined with defined precision under the stated experimental conditions
• 对于平均响应和标准偏差，应进行6 - 10个观察结果

Limit of quantitation = X + (10SD)

• 信号与噪声比应在定量极限下至少为10：1

坚固

坚固measures reproducibility of test results under following conditions :

• 不同实验室在相同条件下为同一样品生成的结果
• 不同分析师在相同条件下为样品生成的结果
• 使用不同仪器的相同分析
• Same analysis under different environmental conditions
• Same analysis using test materials from different sources

坚固

坚固examines the effect of operational parameters changes on the analytical results

• pH
• Temperature
• Operational conditions such as flow rate, injection volume, detection wavelength or mobile phase composition in chromatographic analysis

Variation should be deliberate but within realistic range to study the robustness of the method. The results of the analysis after making the deliberate changes should be within the method’s specified tolerance limits.

Method validationmay appear to be a tedious and time-consuming activity but once the method is validated before adoption, it will not let you down under any circumstances.

What Is the Need for Analytical Method Validation?

在计划对实验室中的样本进行分析之前，您应该了解样本的基本细节，例如其同质性，物理状态，可用数量和稳定性。

也必须了解分析要求，例如：

• Components to be detected and quantified
• Expected concentration levels
• 样品矩阵的性质。换句话说，可能存在的干扰性质可能使确定所需组件的确定复杂化
• Required degree of accuracy and precision
• 适当的分析技术进行分析
• 分析所需的材料和设备
• 所需资源的可用性
• Cost of analysis

上述考虑需要明智地选择该方法。让我们看看您可以访问的那些有用的参考资源。您可以认为自己很幸运，如果您的实验室已经有了可用的验证方法，可以在不采用第二个思想的情况下采用。但是，每次都可能不是这种情况，并且由于以下限制，您会感到需要搜索合适的方法：

• 样品矩阵与常规样品不同，需要不同的提取程序或可以引入不同的干扰
• 所需试剂或玻璃器皿的不可用
• Non-functional or un-calibrated instruments

在这种情况下，您将不得不寻找其他替代方法，因此您必须通过可用选项搜索该方法，例如：

• 标准教科书
• Reference analytical journals
• Pub Med
• 谷歌search

您搜索的次数越多，您可能会更加困惑。让我们假设在浏览参考后，您对特定方法进行了归零。

现在我验证的重要阶段thod before it is put into routine use. You have to keep in mind the fact that the analytical results reported by you can have far-reaching consequences concerning the safety of use of the products, efficacy of drugs, toxic limits for environmental samples, or verdict on legal matters.

As a responsible analyst, you have to make sure that the data reported by you is reliable and would be accepted universally. Such objectives can be achieved only by following the rigorous validation process.

验证becomes necessary:

• 在采用常规使用方法之前
• Whenever there is a requirement of analysis of components in the different sample matrix
• 样本分析需要不同的分析程序

作为常规实践，任何方法都需要在采用前进行验证。但是，在以下情况下需要重新验证它：

• Any changes made in the established procedure
• Any new accessory is added to the existing system
• 该系统经历了已纠正的重大操作问题。

为什么要采用经过验证的分析方法至关重要？

Analytical chemistry is a science which guides you on how to get the desired information from your sample. Themethod validation guidelines提供的

• Selection of appropriate technique
• Procedure to be followed
• 数据分析和解释
• 环境控制和其他要采取的预防措施

您可以认为自己很幸运，如果符合您的分析要求的经过验证的方法已在实验室中可用。或者，您将必须通过文献搜索识别适当的方法。

如果您明确了分析目标，那么您的文献搜索将成功。可以使用Internet搜索引擎或参考文献资源（例如研究期刊）启动搜索。下一步是在采用该方法之前验证该方法。

Benefits of Method Validation

Method validationis a practice which can prove to be beneficial in a number of situations such as:

• 在国际职业会议上的国际声誉或演讲期刊上发布您的发现
• 对自己以及使用您的结果的人灌输一定程度的自尊和信心
• Acceptance of procedures and results by accreditation bodies and external auditors
• Arriving at critical decisions concerning the use of data with regards to the safe use of products by consumers
• Freedom from disputes concerning non-agreement of test results with other laboratories

方法验证在化学分析中的关键作用

Each and every analysis requires the choice of a method that will help you achieve the required objectives. In case an established method is available, you should consider yourself fortunate as this will save you the effort and time required to validate the method before adopting it.

Several options are available to you for the selection of the method to suit your analysis requirements. Such options include a survey of standard analytical textbooks, research journal publications, Google search, etc.

您将在Google搜索中遇到几种方法，但这可能会增加混乱，因为所选方法可能不适用于您的分析要求。

在做出方法选择时，您还必须考虑其他因素，例如：

• Cost of analysis
• 所需试剂和标准的可用性
• 所需工具设施的可用性
• 训练有素的人力的可用性
• Time requirement for analysis
• 方法的简单性

必须记住，最简单的方法可能不是理想的方法，因此您必须仔细评估选择的选择，以查看上面列出的注意事项。

现在，您已经介绍了分析方法验证，请随时在下面的评论中提出任何问题。