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中国药典2015年版9012生物样品定置分析方法验证指导原则一、范围准确测定生物基质（如全血、血清、血浆、尿）中的药物浓度，对于药物和制剂研发非常重要。
这些数据可被用于支持药品的安全性和有效性，或根据毒动学、药动学和生物等效性试验的结果做出关键性决定。
因此，必须完整地验证和记录应用的生物分析方法，以获得可靠的结果。
本指导原则提供生物分析方法验证的要求，也涉及非临床或临床试验样品实际分析的基本要求，以及何时可以使用部分验证或交叉验证，来替代完整验证。
本指导原则二和三主要针对色谱分析方法，四针对配体结合分析方法。
生物样品定量分析方法验证和试验样品分析应符合本指导原则的技术要求。
应该在相应的生物样品分析中遵守G L P原则或GC P原则。
二、生物分析方法验证(一）分析方法的完整验证分析方法验证的主要目的是，证明特定方法对于测定在某种生物基质中分析物浓度的可靠性。
此外，方法验证应采用与试验样品相同的抗凝剂。
一般应对每个新分析方法和新分析物进行完整验证。
当难于获得相同的基质时，可以采用适当基质替代，但要说明理由。
一个生物分析方法的主要特征包括：选择性、定量下限、响应函数和校正范围（标准曲线性能）、准确度、精密度、基质效应、分析物在生物基质以及溶液中储存和处理全过程中的稳定性。
有时可能需要测定多个分析物。
这可能涉及两种不同的药物，也可能涉及一个母体药物及其代谢物，或一个药物的对映体或异构体。
在这些情况下，验证和分析的原则适用于所有涉及的分析物。
对照标准物质在方法验证中，含有分析物对照标准物质的溶液将被加人到空白生物基质中。
此外，色谱方法通常使用适当的内标。
应该从可追溯的来源获得对照标准物质。
应该科学论证对照标准物质的适用性。
分析证书应该确认对照标准物质的纯度，并提供储存条件、失效日期和批号。
对于内标，只要能证明其适用性即可，例如显示该物质本身或其相关的任何杂质不产生干扰。
当在生物分析方法中使用质谱检测时，推荐尽可能使用稳定同位素标记的内标。
9012生物样品定量分析方法验证指导原则9012生物样品定量分析方法验证指导原则是为了确保生物样品定量分析方法的准确性和可靠性而制定的一系列原则和指导。
生物样品定量分析方法验证是指通过实验和数据分析，验证该方法能够准确、可靠地定量分析生物样品中目标物质的含量。
以下是关于9012生物样品定量分析方法验证指导原则的详细介绍。
1.验证目标：明确验证的目标，即要验证的生物样品定量分析方法和目标物质的含量范围。
验证的目标应明确具体，包括检测的目标物质、所用的定量方法和样品类型等。
2.实验设计：合理设计验证实验方案。
验证实验应采用相关的生物样品，并根据目标物质的特性选择合适的样品处理方法。
实验设计应包括重复次数的确定、正负对照样品的设置和实验步骤的详细描述。
3.准确性：验证方法的准确性是指分析结果与真实值之间的一致性。
通过对一定数量的已知浓度样品进行分析，评估方法的准确性。
评估指标可包括回收率、偏差等指标。
4.精密度：验证方法的精密度是指同一样品在重复测量条件下，方法重复性的可靠性。
通过测量同一样品的重复测量值，评估方法的精密度。
评估指标可包括相对标准偏差(RSD)、变异系数等指标。
5.灵敏度：验证方法的灵敏度是指方法对目标物质浓度变化的敏感程度。
通过测量不同浓度样品的分析信号，评估方法的灵敏度。
评估指标可包括最低检测限、定量限等指标。
6.选择性：验证方法的选择性是指方法对其他干扰物质的响应能力。
通过测量其他相关物质的干扰试验，评估方法的选择性。
评估指标可包括干扰物质的峰检出率、峰分离度等指标。
7.稳定性：验证方法的稳定性是指方法在规定条件下的变化范围。
通过存储试样的时效试验、方法的变更试验等，评估方法的稳定性。
评估指标可包括试样的峰面积或峰高百分含量的变化程度。
8.结果分析：根据验证实验的结果，进行数据统计和分析。
根据评估指标的要求，判断方法是否满足准确性、精密度、灵敏度、选择性和稳定性等要求。
结果分析应包括合理的统计方法和结果显示方式。
生物样品定量分析方法验证指导原则1.设计验证计划：验证计划应明确验证的目的、范围和任务，并确定所需资源和时间。
验证计划还应包括验证参数、验证样品数量和测试条件等详细信息。
2.确定验证参数：验证参数是指确定方法准确性、精密度、线性范围、检测限和选择性等方面的参数。
根据具体的分析方法，选择适当的验证参数进行验证。
3.准确性验证：准确性验证包括回收率、加标回收率和对照品比较等方法。
在准确性验证中，使用已知浓度的标准品或对照品进行分析，并比较结果与标准值或真实值之间的差异。
4.精密度验证：精密度验证是通过重复测定同一样品，评估方法的精密度。
重复测定应在相同条件下进行，采用适当的统计方法计算结果的精密度。
5.线性范围验证：线性范围验证涉及不同浓度下的响应与浓度之间的线性关系。
通过分析一系列不同浓度的标准品或对照品，并绘制浓度与响应之间的曲线，确定方法的线性范围。
6.检测限验证：检测限是方法能够可靠检测到的最低浓度。
通过分析一系列浓度低于检测限的样品，并确定能够可靠检测到的最低浓度。
7.选择性验证：选择性验证是评估方法对目标物和干扰物的选择性。
通过分析不同的样品矩阵或添加干扰物，并验证分析结果是否受到干扰。
8.数据分析：在验证完成后，应进行数据分析和评估。
使用适当的统计方法，计算准确度、精密度和线性范围等参数，并评估分析方法的可行性和可靠性。
9.结果报告和文件管理：验证的结果应记录并报告。
应编写验证报告，包括验证计划、实验结果、数据分析和结论等。
还应制定相应的文件管理措施，以确保验证结果的追溯性。
综上所述，生物样品定量分析方法验证是确保分析结果准确性和可靠性的重要步骤。
实施验证需要设计验证计划、确定验证参数、进行准确性、精密度、线性范围、检测限和选择性等方面的验证，并进行数据分析和结果报告。
通过遵循上述指导原则，可以有效验证生物样品定量分析方法的可行性和可靠性。
一、验证目标和范围生物样品定量分析方法的验证目标是评估方法的适用性、准确性、精密度、灵敏度、线性范围、选择性、稳定性和测定限等指标。
验证的范围应包括常规样品、稳定样品和特殊样品等方面。
二、验证计划和报告验证计划应包括验证的目的、范围、方法、样品、检测设备和仪器的选择、验证标准和指标的确定、样品的制备与处理、实验设计和统计分析等内容。
验证报告应包括验证计划的执行情况，具体的验证结果和分析，以及结论和建议等内容。
三、适用性验证方法要考虑分析方法的适用性，包括样品的特性和要求、方法的灵敏度和特异性、样品制备和处理的方法、测定限和线性范围等因素。
四、准确性和精密度准确性和精密度是衡量定量分析方法的重要指标。
准确性的评估包括系统误差的测定和修正，常用方法有标准样品、对比法和加标回收法等。
精密度的评估要考虑到实验操作的稳定性和方法的重复性等因素。
五、选择性和特异性选择性和特异性是评估定量分析方法对目标物质的识别和测定能力的指标。
选择性的评估要考虑方法的干扰物质的影响和实际样品的复杂性。
特异性的评估要考虑到方法对其他物质的干扰以及交叉反应的影响。
六、线性范围和灵敏度线性范围是评估定量分析方法测定结果与目标物质浓度之间的关系的指标。
灵敏度是评估定量分析方法对目标物质低浓度范围内变化的敏感性的指标。
线性范围的评估要包括线性相关系数、回归方程和相关系数的确定。
灵敏度的评估要考虑到检测限和测定限等因素。
七、稳定性稳定性是评估定量分析方法在时间、温度、湿度、光照等条件下的稳定性和耐受性的指标。
稳定性的评估要包括样品的稳定性、试剂的稳定性和仪器的稳定性等方面。
八、测定限测定限是评估定量分析方法可以测定的最低浓度的指标。
测定限的评估要考虑方法的灵敏度和噪声的干扰等因素。
综上所述，中国药典2024年版生物样品定量分析方法验证指导原则（草案）主要涵盖了验证目标和范围、验证计划和报告、适用性、准确性和精密度、选择性和特异性、线性范围和灵敏度、稳定性和测定限等方面的内容。
最新生物样本分析法指导原则为了适应医药工业全球化的进展，生物样本的分析方法也需要全球化的指导原则。
本文就SFDA，FDA及EMA有关生物样本分析的指导原则进行综述分析，了解其中的差异，为研究者进行生物样本分析提供参考。
美国食品药品管理局（FDA）在2001年颁发的关于生物分析方法验证（BMV）的指导原则。
2005年SFDA颁发了三个与生物分析法相关的准则：一是关于非临床研究的，其他两个是关于临床研究的。
最近，欧洲药品管理局（EMA）发布了自己的关于BMV的草案准则[8]。
这些指导原则颁布后，引起了包括EBF（欧洲生物论坛），美国药学科学家协会（AAPS）以及SBDG（上海生物分析讨论组）和BBDG（北京生物分析讨论组）在内的组织的关注。
这些组织都希望建立适用于全球的统一指导原则。
目前这些指导原则存在如下方面的不同：1. 法规遵从与美国FDA的指导原则一样，中国SFDA的指导原则没有明确BMV是否必须遵循GLP规范。
2003年9月以后，SFDA要求，非临床毒代动力学（TK）研究和临床BA/BE研究应分别遵守GLP和GCP 规范，而对于人样本的生物分析没有规定是否仍遵循GLP规范。
EMA 草案明确指出，非临床试验生物样本分析无论是方法学验证还是样本测定均要遵循GLP规范，而人类生物分析研究应遵循GCP原则而不是GLP。
2. 方法验证的范围分析方法学的全部方法学验证的范围，SFDA、FDA和EMA的要求基本一致。
对于在不同种属间进行TK研究的生物样本分析，SFDA 和FDA指导原则要求在更换种属时，仅要求部分方法学验证，而EMA 要求全部验证。
3. 对照和内标SFDA的指导原则要求在分析方法验证时应记录待测物、代谢产物及内标的信息（例如，纯度，来源和稳定性），但目前尚没有具体的质量标准要求。
FDA指导原则规定了待测药物对照品的质量要求，但没有强调作为内标使用的化学品的质量要求[4]。
EMA指导原则既规定了对照品的质量标准，又规定了内标的质量标准，还对内标的稳定性提出了要求[8]。
9012生物样品定量分析方法验证指导原则生物样品定量分析方法验证是确保所采用的分析方法能够准确、可靠地定量生物样品中目标分析物的含量的过程。
验证验证是科学研究中非常重要的步骤，能够确保分析方法的可靠性和有效性。
本文将就生物样品定量分析方法验证的指导原则进行详细介绍。
一、准备工作：1.要明确分析方法的目的和要求，明确分析的目标物质、相关物质和可能影响分析结果的有害物质。
2.正确选择参考标准物质，确保其纯度、稳定性和可获得性。
二、验证方案的制定：1.根据样品的特点和目标物质的性质，制定验证方案。
验证方案应包括验证对象（样品类型、目标物质），验证方法（样品前处理、分析流程），验证指标（准确度、精密度、线性等）等内容。
2.确定验证方案的验证参数，包括样品容量、配制方案、浓度范围等。
三、验证准确度：1.准确度是指分析结果与真实值的接近程度。
验证准确度可以使用加标回收率和对照样品法进行验证。
2.加标回收率验证是在不同浓度水平下，加入一定量的目标物质，并对其进行分析，计算加标回收率。
3.对照样品法是用已知浓度的标准物质进行测定，计算其测定值与已知浓度之间的偏差。
四、验证精密度：1.精密度是指同一样品在同一实验室下，通过多次测定得到的结果的一致性。
验证精密度可以使用重复测定法进行验证。
2.重复测定法是在相同条件下，对同一样品进行多次独立测定，计算测定结果的方差或相对标准偏差。
五、验证线性范围：1.线性范围是指分析方法能够在一定范围内随着目标物质浓度的变化而呈现线性关系。
验证线性范围可以使用系列标准品浓度进行测定，绘制标准曲线。
2.根据标准曲线，分析样品中目标物质的浓度，在线性范围内，测定值与实际浓度之间的误差应在一定范围内。
六、其他验证指标：除了上述的准确度、精密度和线性范围，根据实际需要，还可以对选择性、稳定性、重复性、指标回收率等指标进行验证。
七、结果分析和报告：1.对验证结果进行评价和分析，判断分析方法是否满足要求。
2024药典生物样品定量分析方法验证指导原则2024药典生物样品定量分析方法验证指导原则是指对于药物及其原料药以及相关的生物样品进行定量分析方法验证的准则和指导性原则。
这些原则是为了确保药物和生物样品的准确性、可靠性和重复性，从而保证药物的质量和安全性。
以下是关于2024药典生物样品定量分析方法验证指导原则的详细介绍。
首先，2024药典生物样品定量分析方法验证指导原则要求验证人员具有相应的专业知识和经验，能够进行准确、可靠和重复的测试。
验证人员应具备合适的教育背景、培训经历和实践经验，能够正确操作和解释测试结果。
其次，验证步骤应按照规定的程序和要求进行。
验证步骤包括方法准确性验证、方法精密度验证、方法线性范围验证、方法选择度验证和方法稳定性验证等。
这些步骤能够评估方法的准确性、精密度、线性范围、选择度和稳定性等重要指标。
方法准确性验证是评估方法测量结果与真实值之间差异的步骤。
验证人员可以通过比较方法测得的样品浓度与已知浓度的样品进行对照，以评估方法的准确性。
方法精密度验证是评估方法测量结果的一致性和重复性的步骤。
验证人员应根据规定的程序使用不同的检测设备、分析仪器和操作员对同一样品进行多次测试，以评估方法的精密度。
方法线性范围验证是评估方法在一定浓度范围内能否提供可靠测量结果的步骤。
验证人员应根据规定的程序和要求，测试一系列浓度不同的样品，以评估方法的线性范围。
方法选择度验证是评估方法在存在多种干扰物或类似物质的情况下是否准确测量目标物质的步骤。
验证人员可以通过加入干扰物或其他相关物质来评估方法的选择度。
方法稳定性验证是评估方法在不同条件下是否产生相似结果的步骤。
验证人员应根据规定的程序和要求，测试样品在不同条件下的稳定性，以评估方法的稳定性。
最后，验证人员应根据验证结果制定合适的验证报告，包括验证方案、方法验证结果、数据分析和结论等内容。
验证报告应清晰、准确地描述验证过程和结果，并提供可验证性的依据。
欧洲药典方法验证指导原则简介欧洲药典方法验证指导原则是欧洲药典委员会制定的一套用于验证药品质量的指导原则。
这些原则旨在确保药品在生产和使用过程中的质量和安全性。
本文将详细介绍欧洲药典方法验证指导原则的背景、目的、主要内容以及应用等方面，以便读者对其有更全面和深入的了解。
背景随着现代医学和制药技术的不断发展，对于药品质量和安全性的要求越来越高。
为了确保药品有效性和稳定性，各国纷纷建立了一套严格的质量控制体系。
欧洲药典方法验证指导原则就是其中之一。
目的欧洲药典方法验证指导原则旨在提供一套统一而标准化的方法来验证各种类型的药物。
这些验证方法可以确保生产商能够按照规定进行质量控制，并使监管机构能够评估产品是否符合规定标准。
主要内容1. 方法选择和开发在欧洲药典方法验证指导原则中，首要任务是选择和开发适合的分析方法。
这些方法应该能够准确、可靠地评估药品的质量特性。
2. 方法验证一旦选择和开发了合适的分析方法，就需要对其进行验证。
方法验证是通过实验来证明所选方法可以在一定程度上提供准确和可靠的结果。
3. 方法转移当一个药品生产企业决定采用某种特定的分析方法时，他们需要将该方法从一个实验室转移到另一个实验室。
欧洲药典方法验证指导原则提供了相应的指导原则。
4. 方法更新随着科学技术的不断进步，可能会出现新的分析方法或对现有分析方法进行改进。
因此，欧洲药典委员会会定期审查和更新已有的验证指导原则。
应用欧洲药典方法验证指导原则广泛应用于制药行业和相关领域。
这些指导原则被用于评估各种类型的药物，包括化学药品、生物制剂、植物提取物等。
遵循欧洲药典方法验证指导原则可以帮助制药企业确保其产品的质量和安全性，并与监管机构保持合规。
这些指导原则也有助于促进国际间的药品贸易和技术交流。
结论欧洲药典方法验证指导原则是欧洲药典委员会为了确保药品质量和安全性而制定的一套标准化方法。
这些原则对于制药企业和监管机构来说都是非常重要的。
通过选择、验证和转移合适的分析方法，制药企业可以确保其产品符合规定标准。
生物制品质量控制分析方法验证技术一般原则生物制品质量控制分析方法验证是指确认和验证一种分析方法是否能够得到可靠的测试结果，以确保生物制品的质量符合规定的标准和要求。
验证方法能够确保生物制品的质量控制分析过程的准确性、可靠性和可重复性。
下面将介绍生物制品质量控制分析方法验证的一般原则。
1.验证目标和验证计划：首先需要明确验证方法的目标和验证计划。
验证目标可以是检测方法的准确性、灵敏度、特异性、线性范围、重复性等方面。
验证计划需要明确验收标准、验证样品的选择、验证测试的设计和步骤等。
2.验证样品：验证样品应具备原样品的特性，包括成分、浓度范围等方面。
验证样品需要根据生物制品的特性进行选择，充分覆盖生物制品可能遇到的各种情况。
同时，还需要确定验证样品的数量和配制方法。
3.验证测试的设计和步骤：验证测试应该设计合理，步骤清晰。
验证测试需要包括一系列的实验和分析步骤，以模拟实际生产过程中可能遇到的情况。
验证测试的步骤应包括样品处理、样品测试、数据处理和结果分析等。
4.验证结果的评估：验证测试完成后，需要对结果进行评估和分析。
评估验证结果时，可以使用统计学方法对数据进行分析，比如计算平均值、标准差、相关系数等。
评估结果应与验收标准进行比较，以确定验证方法是否能够得到可靠的测试结果。
5.验证报告的编写：验证测试完成后，需要编写验证报告。
验证报告应包括验证方法的目标、验证样品的选择和配制方法、验证测试的设计和步骤、验证结果的评估和分析等内容。
验证报告需要详细地记录验证过程的每一个步骤和结果，以便审查和审计。
以上是生物制品质量控制分析方法验证的一般原则。
通过验证方法，可以保证生物制品的质量分析过程的准确性和可靠性。
验证报告还可以作为生物制品质量控制分析方法的依据，用于审查和审计。
验证方法的合理设计和严格执行对于保证生物制品的质量至关重要。
人用药品注册技术要求国际协调会ICH协调指导原则生物样品分析方法验证M10草案2019年2月26日授权公开征求意见中在ICH进程第2阶段，ICH大会将相应ICH专家工作组已达成共识的文本或指导原则草案递交给ICH区域监管机构，用以按照相应国家或地区的程序，征求内部和外部意见。
M10指导原则进程法律声明：本指导原则受版权保护，在ICH版权已得到认可的情况下，除ICH标识外，可在公共许可的前提下使用、复制、引用、改编、调整、翻译或传播，在任何情形下需在文件中承认ICH版权。
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ICH协调指导原则生物样品分析方法验证M10ICH 共识指导原则目录1.引言 (6)1.1 目的 (6)1.2 背景 (6)1.3 范围 (6)2.一般原则 (7)2.1 方法开发 (7)2.2 方法验证 (8)2.2.1 完整验证 (8)2.2.2 部分验证 (9)2.2.3 交叉验证 (9)3.色谱法 (10)3.1对照标准品 (10)3.2 验证 (11)3.2.1 选择性 (11)3.2.2 特异性 (12)3.2.3 基质效应 (12)3.2.4 校准曲线和范围 (13)3.2.5 准确度和精密度 (14)3.2.6 残留 (15)3.2.7 稀释完整性 (16)3.2.8 稳定性 (16)3.2.9 重进样重现性 (19)3.3 试验样品分析 (19)3.3.1 分析批 (20)3.3.2 分析批接受标准 (20)3.3.3 校正范围 (22)3.3.4 试验样品重分析 (22)3.3.5 试验样品重进样 (24)3.3.6 色谱图积分 (24)4. 配体结合分析 (24)4.1 主要试剂 (24)4.1.1 标准品 (24)4.1.2 关键试剂 (25)4.2 方法学验证 (25)4.2.1 特异性 (26)4.2.2 选择性 (26)4.2.3 校准曲线和范围 (27)4.2.4 准确度和精密度 (28)4.2.5 残留 (29)4.2.6 稀释线性和钩状效应 (29)4.2.7 稳定性 (30)4.3 试验样品分析 (31)4.3.1 分析批 (31)4.3.2 分析批接受标准 (32)4.3.3 校正范围 (32)4.3.4 试验样品重分析 (33)5.已测样品再分析（ISR） (34)6. 部分验证和交叉验证 (36)6.1 部分验证 (36)6.2 交叉验证 (38)7. 其他考虑事项 (38)7.1 待测物为内源性物质 (38)7.1.1 质控样品 (40)7.1.2 校准曲线 (40)7.1.3 选择性、回收率和基质效应 (40)7.1.4 平行性 (41)7.1.5 准确度和精密度 (41)7.1.6 稳定性 (42)7.2 平行性 (42)7.3 回收率 (42)7.4 最低稀释度 (43)7.5商品化和诊断试剂盒 (43)7.6 新技术或替代技术 (44)7.6.1 干基质方法 (44)8.文档 (45)8.1 摘要信息 (45)8.2 方法验证和生物分析报告文档 (46)9.术语 (50)1.引言1.1 目的本指导原则旨在为化学药物和生物药物定量生物分析方法验证及其在试验样品分析中的应用提供建议，以提高化学药物和生物药物研发和注册申请中生物分析方法验证支持性数据的质量和一致性。
cGMP GCP ISO 17025PIC/S GxP FDA LOD/LOQ OECD QA /QC API EP SOP USP <1225/1226>ICH Q2(R1)分析方法验证基础导论1引言任何分析测试的目的都是为了获得稳定、可靠和准确的数据。
分析方法验证在其中起着极为重要的作用。
方法验证的结果可以用于判断分析结果的质量、可靠性和一致性，这是所有分析质量管理体系不可分割的一部分。
许多法规和质量管理标准也要求实验室进行分析方法验证。
在下列情况下，要求对分析方法进行验证、证实或重新验证：首次用于常规检测前转到另一个实验室时对于验证过的方法，其条件或方法参数发生变化时（例如，仪器性能参数发生改变或样品基质不同时），并且这种变化超出了方法的原适用范围从文献可以看出，行业委员会和监管机构对方法验证相当重视。
本章概述了方法验证对于获得高质量数据有怎样的帮助（还有其他因素，将在以后阐述）。
本书主要讲述实施方法验证的概念和策略。
书中并不详细阐述方法验证的细节。
不过，读者可以找到这方面的各种信息和指南，有研究机构发布的研究成果，也有个人作者发表的文章。
本章对这些文献作一综述。
与分析方法转移以及一些特征参数有关的其他参考文献参见相关章节。
英国政府检测标准集团（The Laboratory of the Government Chemist,LGC ）制定了实验室内部方法验证指南1，其中包含了对实验室认可相关要求的讨论美国食品药品管理局（US FDA ）制定了两个行业指南，分别是分析方法验证2和是生物分析方法验证3ICH 出版了两个方法验证指南。
Q2A 4给出了验证中必须考虑的8种验证参数的术语和定义。
Q2B 5中包含了方法学内容，但是同时声明允许一定的灵活性：“申请人有责任选择最适合其产品的验证步骤和方法。
”IUPAC 6出版了“单个实验室分析方法验证的协调指南”EURACHEM 7出版了方法验证的详细指南。
一、验证目的：
生物样品定量分析方法验证旨在评价一种分析方法在特定条件下，确
定适用对象范围，确定方法的准确性，检测灵敏度和其他相关性能指标的
有效性。
验证的结果可以为方法的合理性提供数据支持，并为方法适用性
评价提供依据。
二、验证的步骤：
三、验证的内容：
生物样品定量分析方法验证的主要内容包括精密度、选定性、准确度、线性范围和检测限等指标的验证。
精密度是指在一定条件下，同一样品的
多次测定结果之间的变异程度，它可以衡量方法的稳定性和重复性；选定
性是指方法能够正确识别和定量分析目标物质，并能排除其他干扰成分的
能力；准确度是指方法能够恢复目标物质真实含量的能力；线性范围是指
方法能够在一定浓度范围内，准确地测定目标物质的含量；检测限是指方
法可以可靠地识别和量化目标物质的最低浓度。
这些指标的验证可以全面
评价生物样品定量分析方法的可靠性和适用性。
四、验证的评价：
对于生物样品定量分析方法验证的评价，应根据验证的目的和内容来
进行判断。
一般来说，验证结果应该符合国际和国家药品标准规定的要求，并能够在实际应用中稳定的使用。
验证结果应该包括实验数据、统计分析
和验证报告等多个方面，并且要进行全面系统的评价，最终确定方法的适
用性。
综上所述，中国药典2024年版生物样品定量分析方法验证指导原则（草案）对于药品质量控制起到了重要的指导作用。
正确的应用和执行该
指导原则，可以有效保证药品的质量和治疗效果，并为药物研发和生产提供科学可靠的方法支持。
生物样品定量分析方法验证指导原则1.方法准确性验证：方法准确性验证是指确定方法的测量结果与真实值之间的偏差程度。
方法准确性可以通过检测已知浓度的标准品进行验证，例如添加已知浓度的化合物到样品中，然后使用待验证的方法测量其浓度。
对于生物样品的定量分析方法，可以使用已知浓度的纯化合物或参考物质进行准确性验证。
2.方法精密度验证：方法精密度验证是指对于同一样品反复进行多次测试，并评估结果的变异程度。
在方法精密度验证的过程中，应采用同一样品的多个并行测试进行比较。
可以计算相对标准偏差（RSD）来评估方法的精密度。
较低的RSD值表明方法具有更好的精密度。
3.方法线性验证：方法线性验证是指在一定范围内检测物质浓度与测量信号之间的线性关系。
为了验证方法的线性，可以制备一系列已知浓度的标准品，并使用待验证的方法进行测量。
然后，可以绘制标准品的浓度与测量信号之间的线性回归曲线，评估曲线拟合程度。
4.方法灵敏度验证：方法灵敏度验证是指确定方法能够检测的最低浓度限制。
可以制备一系列不同浓度的标准品，并使用待验证的方法进行测试。
然后，确定样品最低浓度，使其信号与噪声之间具有足够的信噪比。
5.方法选择性验证：方法选择性验证是指方法能够准确识别和测量目标物质而无需受到其他化合物的干扰程度。
在方法选择性验证的过程中，应添加其他可能存在的干扰物质，并评估它们对目标物质测量的影响。
选择性验证可以通过使用纯化合物或混合物样品进行。
6.方法稳定性验证：方法稳定性验证是指方法在一定时间范围和条件下的测量结果的变化程度。
可以通过在不同时间点和环境条件下进行样品测量，并比较结果来评估方法的稳定性。
稳定性验证可包括样品存储稳定性、制备和处理稳定性、环境因素（如温度、湿度）稳定性等方面的考虑。
总之，生物样品定量分析方法验证是确保方法的可靠性和准确性的重要步骤。
验证过程应包括准确性验证、精密度验证、线性验证、灵敏度验证、选择性验证和稳定性验证。
Guidance for Industry Bioanalytical Methods Validationfor Human StudiesDRAFT GUIDANCEThis guidance document is being distributed for comment purposes only. Comments and suggestions regarding this draft document should be submitted within 60 days of publication in the Federal Register of the notice announcing the availability of the draft guidance. Submit comments to Documents Management Branch (HFA-305), Food and Drug Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD 20852. All comments should be identified with the docket number listed in the notice of availability that publishes in the Federal Register.For questions on the content of the draft document contact Vinod Shah, (301) 594-5635.U.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Drug Evaluation and Research (CDER)December 1998BP #j:\!guidance\2578dft.wpd12/14/98Guidance for Industry Bioanalytical Methods Validationfor Human StudiesAdditional copies are available from:Drug Information Branch (HFD-210)Center for Drug Evaluation and Research (CDER)5600 Fishers Lane, Rockville, MD 20857 (Tel) 301-827-4573Internet at /cder/guidance/index.htmU.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Drug Evaluation and Research (CDER)December 1998BP #j:\!guidance\2578dft.wpd12/14/98Table of ContentsI. INTRODUCTION (1)II. BACKGROUND (1)III. REFERENCE STANDARD (2)IV. PRE-STUDY VALIDATION (3)A. Specificity (3)B. Calibration Curve (4)C. Precision, Accuracy, and Recovery (5)D. Quality Control Samples (6)E. Stability (7)F. Acceptance Criteria (9)V. IN-STUDY VALIDATION (9)VI. DOCUMENTATION (10)REFERENCES (13)j:\!guidance\2578dft.wpd12/14/98 iThis guidance has been prepared by the Biopharmaceutics Coordinating Committee and the Clinical1Pharmacology Section of the Medical Policy Coordinating Committee in the Center for Drug Evaluation and Research (CDER) at the Food and Drug Administration. This guidance document represents the Agency’s current thinking on validation of analytical methods for human studies based on drug or metabolite assay in a biological matrix. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public. An alternative approach may be used if such approach satisfies the requirements of the applicable statute, regulations, or both.j:\!guidance\2578dft.wpd12/14/98GUIDANCE FOR INDUSTRY 1Bioanalytical Methods Validation for Human StudiesI.INTRODUCTIONThis guidance provides assistance to sponsors and applicants of investigational new drug applications (INDs), new drug applications (NDAs), abbreviated new drug applications(ANDAs), and supplements, in developing validation information for bioanalytical methods used in human clinical pharmacology, bioavailability (BA), and bioequivalence (BE) studies. The guidance does not address analytical methods used for nonhuman pharmacology/toxicology studies, CMC information, or in vitro dissolution studies.The information in this guidance is generally applicable to gas chromatography or high-pressure liquid chromatography analytical methods performed on drugs and metabolites obtained from biological matrices such as blood, serum, plasma, or urine. This guidance should also apply to other analytical techniques such as immunological and microbiological methods or otherbiological matrices, such as tissue samples including skin samples, although in these cases a higher degree of variability may be observed.II.BACKGROUNDThis guidance is based primarily on a conference on Analytical Methods Validation:Bioavailability, Bioequivalence and Pharmacokinetic Studies, which was held on December 3 - 5,1990, and sponsored by the American Association of Pharmaceutical Scientists, U.S. Food and Drug Administration, Federation Internationale Pharmaceutique, the Canadian Health Protection Branch, and the Association of Official Analytical Chemists (Shah 1992).Selective and sensitive analytical methods for the quantitative determination of drugs and their metabolites (analytes) are critical for successful performance of clinical pharmacology, BA, and BE studies. Analytical method validation includes all of the procedures recommended todemonstrate that a particular method for the quantitative measurement of an analyte in a given biological matrix, such as blood, plasma, serum, or urine, is reliable and reproducible. The parameters essential to this validation include (1) accuracy, (2) precision, (3) sensitivity, (4) specificity, (5) linearity, and (6) reproducibility. In addition, the stability of the analyte in the matrix under study storage conditions should be determined. Validation involves documenting through the use of specific laboratory investigations that the performance characteristics of the method are suitable and reliable for the intended analytical applications (Shah 1992, Taylor 1983). The acceptability of analytical data corresponds directly to the criteria used to validate the method.Published methods of analyte analysis are often modified to suit the requirements of the laboratory performing the assay. These modifications should be validated to ensure suitable performance of the analytical method. When changes are made to a previously validated method, the analyst should exercise judgment as to how much additional validation is needed. For minor modifications, such as a change in the ratio of solvents for elution, a change in buffer system, the number of extractions of the biological matrix, or a small change in column temperature to obtain better separation, only limited validation may be recommended. For major modifications, such as change of an instrument, solvent system, detector, or temperature, full validation of the modified method should be performed.The analytical laboratory conducting BA and BE studies should closely adhere to FDA’s Good Laboratory Practices (GLPs) (21 CFR Part 58) and to sound principles of quality assurance throughout the testing process. In addition, the analytical methods for in vivo bioavailability studies must meet the criteria in 21 CFR 320.29. The analytical laboratory should have a written set of standard operating procedures (SOPs) to ensure a complete system of quality assurance. The SOPs should cover all aspects of analysis from the time the sample is collected and reaches the laboratory until the results of analysis are reported. They also should include record keeping, security and chain of sample custody (accountability systems that ensure integrity of test articles), sample preparation, and analytical tools, such as methods, reagents, equipment, instrumentation, and procedures for quality control and verification of results.The process by which a specific analytical method is validated may be divided into (1) reference standard preparation, (2) pre-study validation for analytical method development and method establishment, and (3) in-study validation to include study performance, drug analysis, and acceptance criteria (Shah 1992, Brooks 1985). These three processes are described in the following sections of the guidance.III.REFERENCE STANDARDj:\!guidance\2578dft.wpd12/14/982Analysis of drugs and their metabolites in a biological matrix is invariably carried out using samples spiked with calibration standards and quality control (QC) samples. The quality of the reference standard used to prepare spiked samples can affect study data. For this reason, an authenticated analytical reference standard should be used to prepare solutions of known concentrations. If possible, the reference standard should be identical to the analyte. When this is not possible, an established chemical form (free base or acid, salt or ester) of known purity can be used as a surrogate. Three types of reference standards are usually used: (1) certified reference standards (e.g., USP compendial standards); (2) commercially supplied reference standards obtained from a reputable commercial source; and/or (3) other materials of documented purity custom-synthesized by an analytical laboratory or other noncommercial establishment. The source and lot number, certificates of analyses when available, and/or internally or externally generated evidence of identity and purity should be furnished for each reference standard. A master standard (a synthetic batch for which identity and purity are clearly established and acceptable) should be maintained for each reference standard. All subsequently synthesized batches are to be compared chromatographically with that master standard. All reference materials should be checked prior to use to determine if there are significant interfering chromatographic peaks at the retention time of the analyte and/or the internal standard, using the analytical procedure to be used in the study.IV.PRE-STUDY VALIDATIONPre-study validation should include analytical method development and documentation. Validation should be performed for each biological matrix and for each chemical species to be measured in the biological matrix (Shah 1992, Buick 1990). In addition, the stability of quality control samples and the analyte in spiked samples should be determined. Typical performance parameters that should be assessed during pre-study validation include (1) specificity, (2) calibration curve and its linearity, (3) precision, accuracy, recovery, (4) quality control samples,(5) stability of analyte in spiked samples, and (6) acceptance criteria.A.SpecificitySpecificity is the ability of an analytical method to differentiate and quantitate the analyte in the presence of other constituents in the sample and refers directly to the ability of themethod to produce a response for a single analyte (Karnes 1991). For specificity, analyses of blank samples of the appropriate biological matrix (plasma, urine, or other matrix)should be obtained from six individuals under controlled conditions, with reference to time of day, food ingestion, and other factors considered important in the intended study. Each blank sample should be tested for interference using the proposed extraction procedureand chromatographic or spectroscopic conditions. The results should be compared toj:\!guidance\2578dft.wpd12/14/983those obtained with an aqueous solution of the analyte at a concentration near the limit of quantitation (LOQ).Any blank sample with significant interference at the retention time of the drug,metabolites, or internal standard should be rejected. If more than 10% of the blanksamples exhibit significant interference at these retention times, additional matrix blanksamples should be tested. If more than 10% of this subsequent group of blank samplesstill shows interference, the method should be changed to eliminate the interference.Potential interfering substances in a biological matrix include endogenous matrixcomponents, metabolites, decomposition products, and, in the actual study, concomitantmedication. Potential interference from nicotine and common OTC drugs and metabolites, such as caffeine, aspirin, acetaminophen, and ibuprofen should be routinely tested. If the method is intended to quantitate more than one analyte, each analyte should be injectedseparately to determine its retention time and to ensure that impurities from one analyte do not have the same retention time as another analyte.B.Calibration CurveCalibration is the relationship between instrument response and known concentrations of the analyte. A calibration (standard) curve should be generated for each analyte in thesample. A sufficient number of standards should be employed to adequately define therelationship between concentration and response. A calibration curve should be prepared in the same biological matrix as the samples in the intended study by spiking with known concentrations of the analyte. Precautions should be taken to avoid precipitation whilespiking the biological matrix. The number of standards used in constructing a calibration curve will be a function of the anticipated range of analytical values and the nature of the analyte/response relationship. Concentrations of standards should be chosen on the basis of the concentration range expected in a particular study. A calibration curve shouldconsist of a blank sample (matrix sample processed without internal standard), a zerosample (matrix sample processed with internal standard), and five to eight non-zerosamples covering the expected range, including lower LOQ. Blank and standard zerosamples should not be used in the calibration function, but should only serve to evaluateinterference. Additional factors in developing a calibration curve relate to LOQ andlinearity.1.Limit of Quantitation (LOQ)The lowest standard on the calibration curve should be accepted as the limit ofquantitation if the following conditions are met:j:\!guidance\2578dft.wpd12/14/984C No interference present in blanks at the retention time of the analyte at thisconcentration, or typical response at this concentration at least five timesgreater than any interference in blanks at the retention time of the analyteC Analyte peak (response) identifiable, discrete, and reproducible with aprecision of 20% and accuracy of 80-120% (Shah 1992).2.LinearityThe simplest workable regression equation should be used with minimal or noweighting. Selection of weighting and use of a complex regression equationshould be justified. Four factors should be met in developing a calibration curve:C#20% deviation of the LOQ from nominal concentration (Shah 1992)C#15% deviation of standards other than LOQ from nominal concentration (Shah 1992)C At least four out of six non-zero standards meeting the above criteria,including the LOQ and the calibration standard at the highest concentration C0.95 or greater correlation coefficient (r)C.Precision, Accuracy, and RecoveryThe precision of an analytical method describes the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a singlehomogeneous volume of biological matrix. Precision should be measured using aminimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended. The precision determined at each concentration level should not exceed 15% coefficient of variation (CV) except for theLOQ where it should not exceed 20% CV. Precision is further subdivided into within-day, intra-batch precision or reproducibility, which assesses precision during a single analytical run, and between-day, inter-batch precision or reproducibility, which measures precisionwith time and may involve different analysts, equipment, reagents, and laboratories (Shah 1992, USP XXII 1990, Brooks 1985).The accuracy of an analytical method describes the closeness of test results obtained bythe method to the true value of the analyte. Accuracy is determined by replicate analysis of samples containing known amounts of the analyte. A minimum of five determinations j:\!guidance\2578dft.wpd12/14/985per concentration should be conducted for a minimum of three concentrations in the range of expected concentrations. The mean value should be within 15% of the actual valueexcept at LOQ, where it should not deviate by more than 20%. The deviation from thetrue value serves as the measure of accuracy (USP XXII 1990, Brooks 1985).The recovery of an analyte in an assay is the detector response obtained from an amount of the analyte added to and recovered from the biological matrix, compared to thedetector response obtained for the pure authentic standard (Brooks 1985, Mehta 1989).Recovery pertains to the extraction efficiency of an analytical method within the limits of variability. Although recoveries close to 100% are desirable, the extent of recovery of an analyte and/or the internal standard may be as low as 50 to 60% if the recovery is precise, accurate, and reproducible. Recovery experiments should be performed by comparing the analytical results for extracted samples at three concentrations (low, medium, and high)with unextracted standards that represent 100% recovery.D.Quality Control SamplesPre-study validation of an analytical method should be carried out using at least threebatches of biological matrix, where each batch is collected from a different source. Each batch should contain (1) a calibration curve constructed using a blank sample, zerosample, and five to eight non-zero standards, (2) LOQ quality control (QC) samples, (3) low QC samples, (4) medium QC samples, (5) high QC samples, (6) a matrix blanksample, and (7) a reference standard. Quality control samples at concentrations notedbelow should be made from a stock solution separate from that used to prepare thestandards.LOQ QC sample:Same concentration as the lowest non-zero standardLow QC sample:#3 x LOQMedium QC sample:Approximately midway between the high and low QCconcentrationsHigh QC sample:75 to 90% of highest calibration standardThe accuracy of preparation of calibration and QC samples should be checked with thefirst batch. The data from replicate analyses of QC samples and duplicate analyses ofreference standards should be used to obtain the intra-day (within batch) precision, inter-day (between batch) precision, accuracy, and recovery.j:\!guidance\2578dft.wpd12/14/986j:\!guidance\2578dft.wpd12/14/987To obtain within-batch data, the mean, standard deviation, and CV of each QCconcentration in each batch should be calculated. The global (overall) mean, standard deviation, and CV for each QC concentration from the three batches should be calculated to obtain between-batch data. Precision is indicated by the %CVs. Percent accuracy is determined by dividing the mean concentration of a QC by its nominal concentration, and multiplying by 100.E.StabilityDrug stability in a biological fluid is a function of the storage conditions, the chemical properties of the drug, the matrix, and the container system. The stability of an analyte in a particular matrix and container system is relevant only to that matrix and container system and should not be extrapolated to other matrices and container systems. Stability procedures should evaluate the stability of the analytes in biological fluids after long-term (frozen at the intended storage temperature and conditions) and short- term (bench top,room temperature and conditions) storage, and after going through freeze and thaw cycles and the analytical process. The procedure should also include an evaluation of analyte stability in stock solution (Buick 1985, Pachla 1989).All stability determinations should use a set of standard samples prepared from a freshly made stock solution of the analyte in the appropriate analyte-free, interference-free biological matrix. Stock solutions of the analyte for stability evaluation should beprepared in an appropriate solvent at concentrations defined in the method SOP. Further information about validation for these factors appears in the following five sections of the guidance.1.Freeze and Thaw StabilityTesting for freeze and thaw analyte stability should be determined during three freeze and thaw cycles. At least three aliquots at each of the low and highconcentrations should be stored at -20C, or the intended storage temperature, for 024 hours and thawed unassisted at room temperature. When completely thawed,the samples should be transferred back to the original freezer and kept refrozen for 12 to 24 hours. The cycle of thawing and freezing should be repeated two more times, then analyzed on the third cycle. If an analyte is unstable at -20C, the0stability sample should be frozen at -70C during the three freeze and thaw cycles 0(Shah 1992, Buick 1990).2.Short-Term Room Temperature Stabilityj:\!guidance\2578dft.wpd12/14/988Three aliquots of each of the low and high concentrations should be thawed atroom temperature and kept at this temperature from 4 to 24 hours (based on theexpected duration that samples will be maintained at room temperature in theintended study) and analyzed (Buick 1990).3.Long-Term StabilityThe storage time in long-term stability evaluation should exceed the time betweenthe date of first sample collection and the date of last sample analysis. Long-termstability should be determined by storing at least three aliquots of each of the lowand high concentrations under the same conditions as the study samples. Asuggested storage temperature for the majority of drugs and metabolites in a biological matrix is -20C, but lower temperatures (e.g., -70C) may be 0 0recommended to prevent degradation problems observed at higher temperatures.The volume of samples should be sufficient for analysis on three occasions. Theconcentrations of all the stability samples should be compared to the mean of backcalculated values for the standards at the appropriate concentrations from the firstday of long-term stability testing (Buick 1990).4.Stock Solution StabilityThe stability of stock solutions of drug and the internal standard should beevaluated at room temperature for at least 6 hours. The stability samples shouldthen be refrigerated or frozen for 7 to 14 days or other relevant period. Aftercompletion of the desired storage time, the stability should be tested by comparingthe instrument response with that of freshly prepared solutions (Buick 1990).5.Autosampler StabilityThe stability of processed samples in the autosampler should be determined at theautosampler temperature that will be used during analysis, which is usually roomtemperature, but may sometimes be a lower temperature (e.g., when a refrigeratedautosampler is used). Stability should be assessed over the anticipated run time forthe batch size to be used in studies. The stability of both the drug and the internalstandard should be evaluated in validation samples under these conditions bydetermining concentrations on the basis of original calibration standards.Although the traditional approach of comparing analytical results for storedsamples with those for freshly prepared samples has been referred to in thisguidance, other statistical approaches based on confidence limits are also availablefor the development of SOPs for evaluation of an analyte’s stability in a biologicalmatrix (Timm 1985). Whatever approach is used, the SOPs should clearlydescribe the statistical method and rules employed. Additional validation mayinclude investigation of samples from dosed subjects.F.Acceptance CriteriaAn analytical method is considered fully validated when it meets the following criteria:Precision:The between-batch CVs for low, medium, and high concentrations should be #15%, and #20% for the LOQ QC, using a minimum of three batches.Accuracy: The between-batch mean value should be within ±15% of the nominal value at low, medium, and high QC concentrations and should not deviate by more than ±20% at the LOQ.Sensitivity: The lowest standard should be accepted as the limit of quantitation of themethod if the between-batch CV at the LOQ QC is #20%.Specificity: The responses of interfering peaks at the retention time of the analyte should be less than 20% of the response of an LOQ standard. Responses of interfering peaks at the retention time of the internal standard should be #5% of the response of theconcentration of the internal standard to be used in studies.Stability: Long-term, short-term, freeze and thaw, stock solution, and autosamplerstability data should meet the criteria specified in the SOP.V.IN-STUDY VALIDATIONAssays of all samples of an analyte in a biological matrix should be completed within the time period for which stability data are available. In general, analysis of biological samples can be done with a single determination without duplicate or replicate analysis if the assay method has acceptable variability as defined by validation data. This is true for procedures where precision and accuracy variabilities routinely fall within acceptable tolerance limits. For a difficult procedure with a labile analyte, where high precision and accuracy specifications may be difficult to achieve, duplicate or even triplicate analyses may be recommended for better estimate of analyte.j:\!guidance\2578dft.wpd12/14/989A calibration curve should be generated for each analyte to assay samples in each analytical run and it should be used to calculate the concentration of the analyte in the unknown samples in the run. The spiked samples may contain more than one analyte. An analytical run could consist of either all the processed samples to be analyzed as one batch or a batch composed of processed unknown samples of one or more volunteers in a study, QC samples, and calibration standards. The calibration (standard) curve should cover the expected unknown sample concentration range in addition to a calibrator sample at LOQ. Estimation of concentration in unknown samples by extrapolation of standard curves below LOQ or above the highest standard is not recommended. Instead, the standard curve should be redefined or samples with higher concentration should be diluted and assayed (Shah 1992). All study samples from a subject should be analyzed in a single run.Once the analytical method has been validated for routine use, its accuracy and precision should be monitored regularly to ensure that the method continues to work satisfactorily. To achieve this objective, a number of separately prepared QC samples should be analyzed with processed test samples at intervals based on the total number of samples. The QC samples in duplicate at three concentrations (one near the LOQ (i.e., #3 x LOQ), one in midrange, and one close to the high end of the range) should be incorporated in each assay run. The results of the QC samples provide the basis of accepting or rejecting the run. At least four of the six QC samples should be within ±20% of their respective nominal value. Two of the six QC samples may be outside the±20% of their respective nominal value, but not both at the same concentration (Shah 1992, Brooks 1985, Buick 1990, Mehta 1989, Ayers 1981).VI.DOCUMENTATIONThe validity of an analytical method should be established and verified by laboratory studies. Documentation of successful completion of such studies should be provided in the assay validation report. Protocols that define a set of specific directions that must be followed are important if the analytical results are useful for a given purpose.General and specific SOPs and good record keeping are essential parts of a validated analytical method. The analytical protocols and SOPs should be signed and dated by the laboratory director and updated regularly. The SOP should state situations under which reassay of samples is permitted. Reassays should be done in triplicate.The pre-study validation experiments, the data generated from them, and the assay quality control data should be recorded in a bound laboratory notebook. The entries should be signed by the chemist and witnessed by the laboratory supervisor. All records should be available for data audit and inspection.Documentation for pre-study validation should include:! A description of the analytical method! A description of stability studies and supporting data! A description of experiments conducted to determine accuracy, precision, recovery, specificity, linearity, limit of quantitation, and relevant data obtainedfrom these studies!Tables of intra- and inter-day precision and accuracy!Evidence of purity of drug standards, metabolites, and internal standards used in validation experiments!Deviations from SOP, if any, and justification for deviationDocumentation for in-study validation should include:!Calibration curves used in analyzing samples and intra-day accuracy and precision data!Information on inter-day values of QC samples and data on inter-day accuracy and precision from calibration curves and QC samples used for accepting the analyticalrun! A protocol for reassay of samples that describes the reasons for reassay and acceptance criteria for reassayed samples!Reasons for missing samples!Acceptance criteria for reported values when all unknown samples are assayed in duplicate!Deviations from the protocol or SOP, with reasons and justifications for the deviationsDocumentation for submission to the Agency should include:!Pre-study validation data!Calibration curves, equations, and weighting factors used, if any!In-study validation data。
2020生物样品定量分析方法验证指导原则—、范围准确测定生物基质（如全血、血清、血浆、尿）中的药物浓度，对于药物和制剂研发非常重要。
这些数据可被用于支持药品的安全性和有效性，或根据毒动学、药动学和生物等效性试验的结果做出关键性决定。
因此，必须完整地验证和记录应用的生物分析方法，以获得可靠的结果。
本指导原则提供生物分析方法验证的要求，也涉及非临床或临床试验样品实际分析的基本要求，以及何时可以使用部分验证或交叉验证，来替代完整验证。
本指导原则二和三主要针对色谱分析方法，四针对配体结合分析方法。
生物样品定量分析方法验证和试验样品分析应符合本指导原则的技术要求。
应该在相应的生物样品分析中遵守GLP原则或GCP原则。
二、生物分析方法验证（一）分析方法的完整验证分析方法验证的主要目的是，证明特定方法对于测定在某种生物基质中分析物浓度的可靠性。
此外，方法验证应采用与试验样品相同的抗凝剂。
一般应对每个新分析方法和新分析物进行完整验证。
当难于获得相同的基质时，可以采用适当基质替代，但要说明理由。
一个生物分析方法的主要特征包括：选择性、定量下限、响应函数和校正范围（标准曲线性能）、准确度、精密度、基质效应、分析物在生物基质以及溶液中储存和处理全过程中的稳定性。
有时可能需要测定多个分析物。
这可能涉及两种不同的药物，也可能涉及一个母体药物及其代谢物，或一个药物的对映体或异构体。
在这些情况下，验证和分析的原则适用于所有涉及的分析物。
对照标准物质在方法验证中，含有分析物对照标准物质的溶液将被加入到空白生物基质中。
此外，色谱方法通常使用适当的内标。
应该从可追溯的来源获得对照标准物质。
应该科学论证对照标准物质的适用性。
分析证书应该确认对照标准物质的纯度，并提供储存条件、失效日期和批号。
对于内标，只要能证明其适用性即可，例如显示该物质本身或其相关的任何杂质不产生干扰。
当在生物分析方法中使用质谱检测时，推荐尽可能使用稳定同位素标记的内标。
欧盟生物分析方法确证指导原则草案生物分析方法确证指南Guideline on Validation of Bioanalytical MethodsDraft 2009European Medicines Agency1. 前言(背景)2. 范围3. 法律基础4. 方法确证4.1 分析方法的完整确证4.1.1 选择性4.1.2 残留4.1.3 定量下限4.1.4 校正曲线4.1.5 准确度4.1.6 精密度4.1.7 稀释可靠性4.1.8 基质效应4.1.9 稳定性4.2 部分确证4.3 交叉确证4.4 配体结合分析5. 试验样品分析5.1 分析批5.2 分析批的接受标准5.3 校正范围5.4 试验样品再分析5.5 整合6. 已测样品再分析7. 试验报告定义1概要本指南定义了生物样品分析方法的关键因素并提供了建议。
指南集中于药动学样品分析所用分析方法的确证。
此外，也为实际分析试验样品提供了指导。
1. 前言(背景)对于含有新活性物质的药品，以及用于生产线延伸和仿制药开发，测定生物基质中的药物浓度都是重要的。
可能需要这些数据支持新的申请以及已批准药品的变更。
毒动学、药动学和生物等效性试验被用于做出关键性决定，以支持药用物质或药品的安全性和有效性。
因此，很好地表征、完整地确证和记录应用的生物分析方法到一个满意标准是极为重要的。
在特殊情况下，例如分析复杂基质(如固体组织)，当不能满足通常的接受标准时，可能需要使用比本指南定义得更宽接受标准。
这应当说明理由并预先定义。
2. 范围本指南提供生物分析方法确证的要求。
此外，也涉及生物分析方法本身的特定方面，如毒动学试验和临床试验样品的实际分析。
本指南还进一步指出，何时可能使用部分确证或交叉确证，替代一个生物分析方法的完整确证。
14此处不包括某些特殊技术，例如使用C标记药物的放射性标记方法。
但即使在这些情况下，也应该努力应用本指南的原则。
3. 法律基础本指南应结合附录一(指令2001/83修订版)的第一部分和第二部分前言和一般原则来解读。
European Medicines Agency7 Westferry Circus, Canary Wharf, London, E14 4HB, UK1 2 3 London, 19 November 2009Doc. Ref: EMEA/CHMP/EWP/192217/2009COMMITTEE FOR MEDICINAL PRODUCTS FOR HUMAN USE4 (CHMP)5 6 DRAFTGUIDELINE ON VALIDATION OF BIOANALYTICAL METHODS78DRAFT AGREED BY THE EFFICACY WORKING PARTY September 2009 ADOPTION BY CHMP FOR RELEASE FOR CONSULTATION 19 November 2009END OF CONSULTATION (DEADLINE FOR COMMENTS)31 May 20109 Comments should be provided using this template to EWPSecretariat@emea.europa.eu10 KEYWORDSCHMP, EMEA, Guideline, validation, bioanalytical method, analysesGUIDELINE ON VALIDATION OF BIOANALYTICAL METHODS 11121314151617181920212223242526272829303132333435363738TABLE OF CONTENTS1.INTRODUCTION (BACKGROUND) (3)2.SCOPE (3)3.LEGAL BASIS (3)4.METHOD VALIDATION (4)4.1C OMPLETE VALIDATION OF AN ANALYTICAL METHOD (4)4.1.1Selectivity (4)4.1.2Carry-over (5)4.1.3Lower limit of quantitation (5)4.1.4Calibration curve (5)4.1.5Accuracy (6)4.1.6Precision (7)4.1.7Dilution integrity (7)4.1.8Matrix effect (7)4.1.9Stability (8)4.2P ARTIAL VALIDATION (9)4.3C ROSS VALIDATION (9)4.4L IGAND-BINDING ASSAYS (9)5.ANALYSIS OF STUDY SAMPLES (10)5.1A NALYTICAL RUN (11)5.2A CCEPTANCE CRITERIA OF AN ANALYTICAL RUN (11)5.3C ALIBRATION RANGE (12)5.4R EANALYSIS OF STUDY SAMPLES (12)5.5I NTEGRATION (13)6.INCURRED SAMPLES REANALYSIS (13)7.STUDY REPORT (13)DEFINITIONS (16)39404142434546474849505152535456575859606162646566676869707172737475767778 EXECUTIVE SUMMARYThis guideline defines key elements and provides recommendations for the validation of bioanalytical methods. The guideline focuses on the validation of the analytical methods used for pharmacokinetic sample analysis. In addition, guidance will be provided with regard to the actual analysis of study samples.1.INTRODUCTION (background)44Measurement of drug concentrations in biological matrices is an important aspect of medicinal product development for those products containing new active substances as well as for line extensions and generic products. Such data may be required to support new applications as well as variations to authorised drug products. The results of toxicokinetic, pharmacokinetic and bioequivalence studies are used to make critical decisions supporting the safety and efficacy of a medicinal drug substance or product. It is therefore paramount that the applied bioanalytical methods used are well characterised, fully validated and documented to a satisfactory standard in order to yield reliable results.Acceptance criteria wider than those defined in this guideline may need to be used in special situations, such as analysis of complex matrices (e.g. solid tissues), when usual acceptance criteria cannot be met. This should be justified and prospectively defined.2.SCOPE55This guideline provides requirements for the validation of bioanalytical methods.In addition, specific aspects of the bioanalytical method itself will be addressed, e.g. the actual analysis of samples from toxicokinetic studies and clinical trials.Furthermore, this guideline will describe when partial validation or cross validation may represent an appropriate alternative approach to the complete validation of an analytical method.Some special techniques such as radio-labelled analysis methods using 14C labelled drugs, are not covered here, but even in such cases efforts should be made to apply to the principles of this guideline.3.LEGAL BASIS63This guideline has to be read in conjunction with the introduction and general principles (4) and Part I and II of the Annex I to Directive 2001/83 as amended. It applies to Marketing Authorisation Applications for human medicinal products submitted in accordance with the Directive 2001/83/EC as amended, and Regulation (EC) No. 726/2004, in which the analysis of drug concentrations in a biological matrix is part of the application.The validation of bioanalytical methods and the analysis of study samples should be performed in accordance with the principles of Good Laboratory Practice (GLP). However, as human bioanalytical studies fall outside of the scope of GLP, as defined in Directive 2004/10/EC, the sites conducting the human studies are not required to be monitored as part of a national GLP compliance programme. In addition, for clinical trials in humans the principles of Good Clinical Practice (GCP) should be followed.Furthermore, reference is made to the following EMEA guidelines:•Note for guidance on good clinical practices (CPMP/ICH/135/95).•Note for guidance on validation of analytical procedures: text and methodology(CPMP/ICH/381/95).81828384858687888990919293949596979899100 101 102103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 119 120 121 4.METHOD VALIDATION794.1Complete validation of an analytical method80A complete method validation should be performed for any analytical method whether new or basedupon literature.The main objective of method validation is to demonstrate the reliability of a particular method for the determination of an analyte concentration in a specific biological matrix, such as blood, plasma, urine, saliva or tissue. Moreover, validation should be performed using the same anticoagulant as for the study samples. A full validation should be performed for each species concerned.In some cases, it may be problematic for validation purposes to obtain an identical matrix compared to the matrix of the study samples. A suitable alternative matrix may be used, e.g. synthetically prepared cerebrospinal fluid, if justified.The main characteristics of a bioanalytical method that are essential to ensure the acceptability of the performance and the reliability of analytical results are: selectivity, lower limit of quantitation, the response function (calibration curve performance), accuracy, precision, matrix effects, stability of the analyte(s) and any internal standard in the biological matrix and the stock and working solutions under the entire period of storage and processing conditions.Usually one analyte or drug has to be determined, but on occasions it may be appropriate to measure more than one analyte. This may involve two different drugs, but can also involve a parent drug with its metabolites, or the enantiomers or isomers of a drug. In these cases the principles of validation and analysis apply to all analytes of interest.Reference standardsDuring method validation, a blank biological matrix will be spiked with the analyte of interest using solutions of reference standard. In addition, an internal standard (IS) is normally used in chromatographic methods.It is important that the quality of the reference standard and IS is ensured, as the quality (purity) may affect the outcome of the analysis, and therefore the outcome of the study data. Therefore the reference standards used for the analytical validation and analysis should be obtained from an authentic and traceable source. Suitable reference standards, include certified standards such as compendial standards (EPCRS, USP, WHO), commercial available standards, or fully characterised standards prepared in-house or by an external non-commercial organisation. Suitability of the reference standard should be scientifically justified. The use of certified standards is not needed for IS, as long as the suitability for use is demonstrated, e.g. lack of interference is shown for the substance itself or any impurities thereof.Whoever the supplier, a certificate of analysis is required to ensure quality, stability, storage conditions, expiration date, batch number and purity of the reference standards.When MS detection is used in the bioanalytical method, a stable isotope-labelled IS is recommended to be used whenever possible. However, it is essential that the labelled standard is of the highest isotope purity and that no isotope exchange reaction occurs. The presence of any unlabelled analyte would otherwise introduce a bias in the results.4.1.1Selectivity118The analytical method should be able to differentiate the analyte(s) of interest and IS from endogenous components in the matrix (i.e. blood, plasma, urine) or other components in the sample. Selectivity should be proven by using at least 6 sources of the appropriate blank matrix, which are individually122 123124 125 126 127128 129 130 131 132 133 134 135 136 137 138 139 141 142 143 144 145 146 147148 149 151 152 153155 156 157 158 159 160 161 162 163 164 165 166 analysed and evaluated for interference. Absence of interfering components is accepted where the response is less than 20% of the lower limit of quantitation for the analyte.It may also be necessary to investigate the extent of any interference caused by metabolites of the drug(s), interference from degradation products formed during sample preparation, and interference from possible co-administered medications. Co-medications normally used in the subject population studied should be taken into account.The possibility of back-conversion of a metabolite into parent analyte during the successive steps of the analysis (including extraction procedures) should also be evaluated, when relevant (e.g. acidic metabolites to ester, unstable N-oxides or glucuronide metabolites, lactone-ring structures). Preferably, blank matrix (and/or samples spiked with analyte at a concentration not higher than 3 times the lower limit of quantitation) should be spiked with concentrations of the metabolite of interest, representing the actual highest in vivo metabolite concentrations, the sample should be processed, and the chromatogram should be evaluated for the formation of the parent analyte. The extent of back-conversion should be established and the impact on the study results discussed. It is acknowledged that this evaluation will not be possible early during drug development of a new chemical entity when the metabolism is not yet evaluated. However, it is expected that this issue is taken into account and the analytical method is revalidated as further knowledge regarding metabolism of the active substance is gained during drug development.4.1.2Carry-over140Carry-over should be addressed and minimised during method development. Carry-over may not affect accuracy and precision (see section 4.1.5 and 4.1.6). During validation carry-over should be assessed by injecting blank samples after a high concentration sample or calibration standard. If it appears that carry-over is unavoidable, specific measures should be considered, tested during the validation and applied during the analysis of the study samples. This could include the injection of blank samples after samples with an expected high concentration, before the analysis of the next study sample.Randomisation of samples should be avoided, as this may interfere with the detection and assessment of carryover problems.4.1.3Lower limit of quantitation150The lower limit of quantitation (LLOQ) is the lowest amount of analyte in a sample which can be quantified reliably, with an acceptable accuracy and precision (see Accuracy and Precision). The LLOQ should be adapted to expected concentrations and to the aim of the study.4.1.4Calibration curve154The response of the instrument with regard to the analyte should be known, and should be evaluated over a specified concentration range. The concentrations to be analysed (calibration standards) should be prepared in the same matrix as the matrix of the intended study samples by spiking the blank matrix with known concentrations of the analyte (and IS). There should be one calibration curve for each analyte studied in the method validation and for each analytical run.Before carrying out the validation of the analytical method it should be known what concentration range is expected. This range should be covered by the calibration curve range, defined by the LLOQ being the lowest calibration standard and the upper limit of quantitation (ULOQ), being the highest calibration standard. The range should be justified based on scientific information.A minimum of six calibration concentration levels should be used, excluding the blank sample(processed matrix sample without analyte and without IS) and a zero sample (processed matrix with IS).167 168 169170 171 172 173174 175 176 177 178179 180 181 183 184 185 186 187 188 189 190191 192 193 194 195 196 197 198 199200 201 202 203 204205 206 207 208 A relationship which can simply and adequately describe the response of the instrument with regard to the analyte should be applied. The blank and zero samples should not be taken into consideration to calculate the calibration curve parameters.The calibration curve parameters should be submitted (slope and intercept in case of linear fit). In addition, the back calculated concentrations of the calibration standards should be presented together with the calculated mean accuracy values (see definition of Accuracy below). At least 3 calibration curves should be evaluated.The back calculated concentrations of the calibration standards should be within ±15% of the nominal value, except for the LLOQ for which it should be within ±20%. At least 75% of the calibration standards with a minimum of six, must fulfil this criterion. In case a calibration standard does not comply with these criteria, this calibration standard sample should be rejected, and the calibration curve without this calibration standard should be re-evaluated, including regression analysis. Although it may be clear from stability data that the analyte is sufficiently stable in the matrix of interest, it is recommended that freshly prepared calibration curves are used during validation of the bioanalytical method.4.1.5Accuracy182The accuracy of an analytical method describes the closeness of the determined value obtained by the method to the true concentration of the analyte (expressed in percentage). Accuracy should be assessed on samples spiked with known amounts of the analyte, the quality control samples (QC samples).During method validation accuracy should be determined by replicate analysis using a minimum of5 determinations at a minimum of 4 concentration levels which are covering the calibration curverange: the LLOQ, within three times the LLOQ (low QC), around 50% of the calibration curve range (medium QC), and at about 75% of the upper calibration curve range (high QC).The QC samples are analysed against the calibration curve, and the obtained concentrations are compared with the nominal value. The accuracy should be reported as percent of the nominal value.Accuracy should be evaluated for the values of the QC samples obtained within a single run (the within run accuracy) and in different runs (the between-run accuracy). The latter will support the accuracy over time.To enable evaluation of any trends over time within one run, it is recommended to demonstrate accuracy of QC samples over at least one of the runs with a size equivalent to a prospective analytical run.Within-run accuracyFor the validation of the within-run accuracy, there should be a minimum of five samples per concentration level at LLOQ, low, medium and high QC samples in a single run. The mean accuracy value should be within 15% of the nominal values for the QC samples, except for the LLOQ which should be within 20% of the nominal value.Between –run accuracyFor the validation of the between-run accuracy at least five determinations per concentration per run at LLOQ, low, medium and high QC samples from three runs analysed on at least two different days should be evaluated. The mean accuracy value should be within 15% of the nominal values for the QC samples, except for the LLOQ which should be within 20% of the nominal value.209 210 211213 214 215 216 217 218 219 220 221 222 223225 226 227 228 229 231 232 233 234 235 236 237 238239 240 241 242 243 244 245 246 247 248 249 Reported method validation data and the determination of accuracy and precision should include all outliers; however, calculations of accuracy and precision excluding values that are statistically determined as outliers should additionally be reported.4.1.6Precision212The precision of the analytical method describes the closeness of repeated individual measures of analyte. Precision is expressed as the coefficient of variation (CV). The statistical method for estimation of the precision should be predefined and calculated according standard practise. Precision should be demonstrated for the LLOQ, low, medium and high QC samples, within a single run and between different runs, i.e. using the results generated for demonstration of accuracy.Within-run precisionThe within-run CV value should not exceed 15% for the QC samples, except for the LLOQ which should not exceed 20%.Between –run precisionThe between-run CV value should not exceed 15% for the QC samples, except for the LLOQ which should not exceed 20%.4.1.7Dilution integrity224Dilution of samples should not interfere with the accuracy and precision. Dilution integrity should be demonstrated by spiking the matrix with an analyte concentration above the ULOQ and dilution of this sample with blank matrix (at least five determinations per dilution factor). Accuracy and precision should be within the set criteria, i.e. within ±15%. Dilution integrity should cover the applied dilution of the study samples.4.1.8Matrix effect230Matrix effects should be investigated when using mass spectrometric methods, using at least 6 lots of matrix including haemolysed, hyperlipidaemic and if applicable, sample matrix from special populations, such as renally or hepatically impaired populations.For each analyte and the internal standard, the matrix factor (MF) should be calculated in each lot of matrix, by calculating the ratio of the peak area in the presence of matrix (measured by analysing blank matrix spiked with analyte at a concentration of maximum 3 times the LLOQ after extraction), to the peak area in absence of matrix (pure solution of the analyte). The IS normalised MF should also be calculated by dividing the MF of the analyte by the MF of the IS.The CV of the IS-normalised MF calculated from the 6 batches of matrix should not be greater than15 %.If this approach cannot be used, for instance in the case of on-line sample preparation, the variability of the response from batch to batch should be assessed by analysing at least 6 batches of matrix in triplicate, spiked at a concentration of a maximum of 3 times the LLOQ. The validation report should include the peak areas of the analyte and of the IS and the calculated concentration for each individual sample. The overall CV calculated for the concentration should not be greater than 15 %. The mean concentration should be within 15 % of the nominal concentration. The mean concentration should also be reported for each individual batch of matrix; a deviation of this mean from the nominal concentration of more than 20 % in any individual batch of matrix should lead to additional investigations.250 251252 253 254 255 256 258 259 260 261262 263 264 265 266 267 268 269 270 271272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 If the matrix is difficult to obtain, less than 6 different batches of matrix may be used, but this should be justified. However, matrix effects should still be investigated.If a formulation for injection to be administered to the subjects or animals contains excipients known to be responsible for matrix effects, for instance polyethylene glycol or polysorbate, matrix effects should be studied with matrix containing these excipients, in addition to blank matrix. The matrix used for this evaluation should be obtained from subjects or animals administered the excipient, unless it has been demonstrated that the excipient is not metabolised or transformed in-vivo.4.1.9Stability257Evaluation of the stability should be carried out to ensure that every step taken during sample preparation and sample analysis, as well as the storage conditions used do not affect the concentration of the analyte. Any deviation from the initial concentration that does occur must be within acceptable limits.Stability should be ensured for every step in the analytical method, meaning that the conditions applied to the stability tests, such as sample matrix, materials storage and analytical conditions should be similar to those used for the actual study samples. Stability cannot be proven by literature data.Stability of the analyte and IS in the studied matrix is evaluated using at least triplicates samples of the low and high QC samples which are analysed immediately after preparation and after the applied storage conditions that are to be evaluated. The QC samples are analysed against a calibration curve, obtained from freshly prepared calibration standards, and the obtained concentrations are compared to the nominal concentrations. The deviation should be within ±15%.Stability of the stock and working solutions should be tested with an appropriate dilution, taking into consideration the linearity and measuring range of the detector.Stability studies should investigate different storage conditions over time periods that equal or exceed those applied to the actual study samples.Normally, as an example, the following stability tests should be evaluated:•stock solution and working solution stability,•freeze and thaw stability of the analyte in the matrix from freezer storage conditions to room temperature,•stability of the analyte in matrix stored in the refrigerator, if applicable,•bench top stability of the analyte in matrix at room temperature,•long term stability of the analyte in matrix stored in the freezer at the same storage temperature as the study samples,•bench top stability of the processed sample at room temperature or under the storage conditions to be used during the study (dry extract or in the injection phase), if applicable, •on-instrument/ autosampler stability of the processed sample at injector or autosampler temperature.Regarding the freeze and thaw stability: The QC samples are stored and frozen in the freezer at the intended temperature and thereafter thawed at room temperature. After thawing, samples are refrozen again applying the same conditions. At each cycle, samples should be frozen for at least 12 hours289 290 291 292 293 294 295 296 297 298 299301 302 303 304 305 306 307 309 310 311 312 313 314 315 316318 319 320 321 322 323324 325 326 327 328 329 330 before they are thawed. The number of cycles in the freeze-thaw stability should equal or exceed that of the freeze/thaw cycles of study samples.Regarding long term stability of the analyte in matrix stored in the freezer: The QC samples should be stored in the freezer under the same storage conditions and at least for the same duration as the study samples. For the evaluation of the long term stability it is not acceptable to use study samples, as the nominal concentration is unknown, and can therefore not be used as reference. It is recommended that evaluation of long term stability is carried out before the start of the actual study.Sufficient attention should be paid to the stability of the analyte in the sampled matrix directly after blood sampling of subjects and further preparation before storage, to ensure that the obtained concentrations by the analytical method reflect the concentrations of the analyte in the subject at the moment of sampling..4.2Partial validation300In situation where minor changes are made to an analytical method that has already been validated, a full validation may not be necessary, depending onto the nature of the applied changes. Changes for which a partial validation may be needed include transfer of the bioanalytical method to another laboratory, change in equipment, calibration concentration range, storage conditions etc. All modifications should be reported and the scope of revalidation or partial validation justified.In most cases, provision of additional accuracy and precision data or relevant additional stability data on the modified issue may be sufficient.4.3Cross validation308Where data are obtained from different study sites, comparison of those data is needed, and a cross validation of the applied analytical methods should be carried out. Differences in sample preparation or the use of another analytical method may result in different outcomes between the study sites. Cross validation should be performed in advance of study samples being analysed if possible. For the cross validation, the same set of QC samples should be analysed by both analytical methods. The outcome of the cross validation is critical in determining whether the obtained data are reliable and whether they can be compared and used. The difference between the two measurements should not exceed 15%.4.4Ligand-binding assays317Ligand-binding assays or immunoassays are nowadays especially used for macromolecules. In these assays, quantification is based on macromolecular interactions between the macromolecule and antibody.The validation principles and the issues with regard to analysis of study samples as indicated before, should also be applied in general for ligand-binding assays. However, the following issues need special attention.One of the issues is the fact that macromolecules tend to be heterogeneous (small differences in for instance glycosylation, or phosphorylation). Therefore validation must cover issues such as batch-to-batch differences in glycosylation and metabolic differences in phosphorylation.In addition, macromolecules may be structurally comparable to endogeneous compounds. Therefore, specificity of the antibody and selectivity of the assay are critical parameters for ligand-binding assays.Specificity of an antibody refers to its ability to mainly bind the antigen of interest. Ideally the antibody should be specific such that no cross-reactivity with structural related compounds occurs.331 332 333 334 335 336 337 338 339 340341 342 343 344345 346 347 348 349350 351 352 353 354355 356357 358 359 360 361362 363 364365366 367 368 369 371 372 373 374 Specificity is validated by using at least 10 sources of sample matrix, spiked at or near the LLOQ. The presence of endogenous antibodies to the analyte may interfere with the analysis and should be taken into account. As interference may be concentration dependent, it would be helpful to estimate the concentration below which interference occurs. Due to interference of endogenous compounds, the blank response may exceed 20% of the LLOQ, however this may be acceptable, as long as it does not affect accuracy. If a surrogate matrix is used for validation, the comparability with regard to study sample matrix should be demonstrated, because of the disease state of a subject, the study matrix may contain different components that may interfere. It is important that the sample matrix and the standard matrix are equivalent. If not, it must be shown that the dose-response relation (e.g. the slope and asymptotes of a four-parameter curve) is unaffected by the matrix.Reference standards should be selected in such a manner that specificity is ensured, and binding characteristics are durable and stable regarding antibody/antigen complex formation. A change in reference standard during analysis may affect this, and suitability of the new reference standard should thus be demonstrated.With regard to the calibration curve, the response is measured indirectly and as a result the response function is in most cases nonlinear. Moreover, generally the calibration curve range is limited (<2 orders of magnitude). In case anchor calibration standards are used, the additive value should be clearly documented. Accuracy of the back calculated concentrations should be within ±20%, except for LLOQ and ULOQ which should not exceed 25%.At least three QC samples should be included (low, medium and high) in each analytical run. The within-run and the between-run accuracy and precision should be within ±20%. Furthermore, the total error (sum of the absolute value of mean accuracy and precision should be less than 30% (40% at the LLOQ and ULOQ). The results of all runs should be included for validation, except in cases where errors are obvious and documented.It is recommended that the size of the run during validation is comparable to that of a run during analysis of study samples (or visa versa).It should be demonstrated that characteristics of the analyte are not affected by the methods of sample preparation, additives (e.g. anticoagulants), and stability during the whole process. Evaluation should not only address chemical and physical properties, but also biological integrity (i.e. maintenance of antibody binding affinity). In addition, this also accounts for changes after validation, e.g. a change in used diluents or reagents during analysis of study samples.If for analysis of study samples microtiter plate-based assays are used, a batch may comprise several individual plates, but each plate should contain an individual set of calibration standards and QC samples, to compensate for difference in plate performance.Commercial kitsCommercial kits may have been developed for other use or purposes than the intended use, e.g. analysis of samples obtained in bioequivalence studies. Therefore, commercial kits need to be re-validated, meaning that the LOQ and the QC samples in the actual concentration range perform accurately and precisely. The principles of validation listed before apply.5.ANALYSIS OF STUDY SAMPLES370After complete validation of the analytical method, analysis of study or subject samples may be carried out. Depending on the time period between validation and the analysis of the study samples it may be necessary to verify the performance of the method before start of the analysis of study samples.。
