手动积分的利和弊！

繁华洛尽

手动积分的利和弊！
来源：OurGMP


　　实验室数据完整性已成FDA检查制药企业的一把利器，其中一条关于随意手动积分或者无书面程序规定手动积分，说明了FDA本身并不是不认可手动积分，而是要求有操作规程规定什么情况可以手动积分，如何积分，并且应保存并记录积分参数。


　　CFDA紧跟FDA的步伐，在2016年度多次飞检中，开具多条数据完整性方面的缺陷，收回多张GMP证书。关于手动积分并没有开具缺陷，制药企业应防患于未然，提前把手动积分管理好。


　　一、关于手动积分的指南


　　关于手动积分，并没有指南给出明确的定义。笔者只查到有两篇生物样本分析的方法验证指南规定了再积分(Reintegration)的注意事项。


　　FDA的2001年定稿指南 Bioanalytical Method Validation，及相应的2013年草案指南也有类似的要求。 生物样本分析的方法验证2013草案指南，第III.C节“样本数据再积分”规定：SOP应该确定再积分的标准，和怎样进行再积分;应清楚描述、并记录再积分的理由;应报告原始和再积分数据。第VII.D节还规定：再积分数据应有管理人员授权再积分。


　　EMA的2011年指南 Guideline on bioanalytical method validation，第5.5节规定：应该有SOP描述色谱的积分和再积分。应在分析报告中讨论偏离这个SOP的任何偏差。应该记录色谱积分、再积分的积分参数、初始积分数据、最终积分数据，并在要求时立即可得。


　　二、什么情况下可以进行手动积分?


　　1)低分离度或低响应;


　　2)流动相不稳定，基线紊乱，保留时间变化小;


　　3)运行过程中，出现异常峰;


　　4)软件积分的局限性，如由于软件参数阈值的设置导致峰未积分，峰起点和终点间出现肩峰和异常的基线漂移，软件错误(峰未积分和错误识别峰);


　　5)由于样品母体的干扰导致的复杂色谱图：裂峰、目标杂质的同时洗脱/肩峰、基线噪音、负峰、基线上升或下降(由于某些梯度程序)、峰的严重拖尾、烃类的存在导致自动积分不正确。


　　三、手动积分的权限及操作


　　操作员在数据系统自动积分不正确时，领取手动积分处理报告表并填写，手动积分申请被QC经理批准后由QC经理或QC组长(若有)或其指定人员(一般是指经验丰富的资深QC)方可进行手动积分。


　　四、手动积分注意事项


　　1)手动积分不能试图通过以下动作来使数据符合标准要求：减少峰(减少峰面积);增加峰(增加峰面积);改变峰高;


　　2)同一次检验的所有标准品、工作对照品、样品等必须使用同一方法进行手动积分。


　　3)手动积分结束后，应打印自动积分图谱和手动积分图谱附于手动积分申请表之后，提交QC经理审核;QC经理应复核完整的数据找出根因，基于根因制定相应的CAPA措施来避免类似事件。


　　4)自动积分的图谱不得删除或被覆盖，应与手动积分图谱保存于计算机中;打印的自动积分图谱和手动积分图谱应标识清楚签名，随同手动积分申请表附于检验原始记录后面一起交质量管理部经理、QP审核无问题后归档。


　　五、色谱软件选型、权限设置和结果显示


　　满足合规要求的色谱软件(例如Waters和Angilent的软件)，能够分别设置不同权限，例如普通化验员不具备进行手动积分的权限，需要主管进行操作，有些软件则不具备这些功能;即使是同一个品牌的软件，也有不同版本;即使软件有这个功能，企业也不一定启用。


　　有两个信息是否显示，对色谱数据透明度非常重要：方法名称和数据版本。如果企业不使用手动积分(重画基线)，而采用修改参数的方式，则每修改一次，是不同的方法(规范的管理程序会要求更改方法名称保存，否则就只能到审计追踪才能查出来这个方法已经被修改了)，而打印的色谱图可以选择是否显示方法; 每处理一次数据，会形成一个数据版本，同样的，打印的色谱图可以选择是否显示数据版本。一个完全透明的结果显示，会在打印的色谱图上显示“方法名称+数据版本”(同时要求修改参数则改变方法名称，不需要QA或检查员仔细检查审计追踪才能发现修改痕迹)。


　　What is Manual Integration?


　　什么是手动积分?


　　So far we have discussed the regulatory issues around peak integration, the main integration parameters, and the three rules of integration so that you have a fighting chance for automatically integrating your peaks and there is no need to intercede manually. However, we now need to turn our attention to manual integration. As we can see from the regulatory citations and the bioanalytical guidance documents, what is needed is an SOP to control, manage, and authorize integration — both automatic and manual.我们已经讨论了关于峰积分发布的相关法规，主要的积分参数，和重新积分的三个原则，满足这些我们能对峰进行自动积分而没必要进行手动积分。但是，现在我们需要把注意力转向手动积分。正如我们从法规引文和生物分析指南文件看到的，我们需要的是控制、管理批准自动/手动积分的SOP。 In an ideal world there would be a definition of manual integration. However, there is not a definition available for manual integration that I could find. Neither in Dyson, nor on any regulatory website. This was the point made by Hill et al. (7), who discussed the scope and terminology of manual reintegration and they concluded that it is essential to develop a consensus with respect to definitions. The problem, they noted, is that that a consensus does not exist.手动积分的定义应该确定。但是我没有找到一个可以使用的关于手动积分的定义。无论是Dyson还是在所有法规网站中都没有。Hill et al. (7) 中提到过，他讨论了手动积分的范围和专业术语，他们都说有必要对其定义进行统一。问题是，没有统一。 This raises an important question: if we can’t define manual integration how can we write an SOP on integration as a whole?这就出现了一个重要问题：如果我们不能定义手动积分，怎样在整体上编写一个关于积分的SOP? Scope of an Integration SOP 积分SOP的范围?


　　In the spirit of the early pioneers, this column will present a possible approach to writing an SOP in the form of a flowchart that will consider some, but not all, options for both automatic and manual integration. Of course, there is a little trepidation because one definition of pioneer is finding new and exotic ways to die (!) However, I hope that this column and my views stimulate a debate about what constitutes manual integration and we can get a definition agreed upon.本着拓荒者的精神，这个专栏将提出一个可能的方法来编写SOP，以流程图的形式讨论了手动积分和自动积分的一些选项。当然，这有一些担忧，因为拓荒就是作死的节奏啊。但是，我希望这个专栏和我的观点能够引发一个关于手动积分的大讨论，使我们能够得到一个统一的定义。 In this discussion we need to balance sound science with regulatory compliance. Therefore, let me pose a question — which is worse: not reintegrating when you know the results are wrong or accepting wrong results when you can see an unknown peak in the sample chromatograms? You see the dilemma? We know the outcomes of both situations are undesirable, but as a result of inflexible procedures or fear of the regulations sound scientific judgement cannot be exercised.在这个讨论中我们需要平衡各种科学合理的法规符合性。因此，让我们提出一个问题看看一下哪一个更糟糕：当你知道结果错误的时候没有重新积分，或者你看到样品色谱图中有未知峰，然而你接受了错误的结果?你看到困境了吗?我们知道这些情况是不可取的，但是死板的程序或者对法规的担忧使得科学判断不能行驶。 First, let us consider a “no manual integration allowed” option. Personally, I think that this is an untenable situation, particularly if a regulated laboratory is using recently developed methods, analyzing impurities where the limits of quantification or detection are close to baseline noise, or investigating stability of products. In these situations, manual reintegration is scientifically sound and defendable provided that it is covered in the integration SOP.首先，让我思考一个“不允许手动积分”的观点，个人认为这是一个站不住脚的观点，特别是受控的实验室使用近期开发的方法分析杂质，且定量限或检测限接近基线噪音，或者在研究产品的稳定性时。这些情况手动积分都是科学合理的并且在积分SOP中可以涵盖的。 A suggested flowchart for consideration of integration is shown in Figure 3. It begins with the completion of the chromatographic run and the automatic integration of peaks by the original processing method. The resulting chromatograms are reviewed by the chromatographer to see if retention times and peak shapes are as expected, the peak(s) have been correctly identified, baseline placement is as required by the analytical procedure, and the sample is integrated consistently with the standard. There may be other criteria that an individual laboratory wishes to apply. We now come to a decision point: is the run acceptable? If yes the individual results and the reportable value are calculated and all is well. All data at this point have been calculated automatically by the CDS, the chromatographer is merely confirming what the software has done conforms to pre-defined expectations.在图3是关于积分考虑的建议性流程图。它在色谱运行完成时开始，通过初始处理方法进行峰的自动积分。色谱工作人员对色谱结果进行复核：保留时间和峰的性状都是否和预期一致?峰是否能够被正确鉴别?基线位置是否符合分析程序?样品按照标准进行积分?个别实验室可能有其他标准。现在我们需要决策：运行是否可接受?如果是，就计算各个的结果和报告的数值，一切都好。所有数据都通过CDS进行自动计算，色谱工作人员仅需要确认软件所做的符合预期期望就可以了。 However, if the integration is not acceptable we move to a second decision point: is manual integration (whatever that term may cover) permitted for this analytical procedure? If not the next stage is a laboratory investigation. What methods could we consider for inclusion for no manual integration? Perhaps measurement of active pharmaceutical ingredients (APIs) or registered methods for finished product for the active ingredient? If these peaks cannot be integrated correctly are you out of control? We will consider if this is tenable when we have finished discussing the flowchart.但是，如果积分不能接受我们需要进行第二个决策：该分析程序是否允许手动积分(无论该术语包含哪些内容)?如果不能，下一阶段就是实验室调查。我们应该考虑使用哪些方法不能手动积分吗?如果是APIs(active pharmaceutical ingredients)的检测或对最终产品的活性成分的注册方法呢?如果这些峰不能被正确积分，你就没辙吗?我们将在完成这个流程图讨论的时候思考这是否站得住脚? In my view, manual integration must be specifically prohibited in the following circumstances:我认为，在以下情况手动积分是禁止使用的。


　　Symmetrical peaks that have acceptable baseline to baseline fitting following automatic integration.有可以接受基线的对称峰，基线拟合后可以自动积分。


　　Enhancing or shaving peak areas to meet SST acceptance criteria or allowing a run to meet the test specification.增加或消减峰面积以符合SST(适用性试验)接受标准或者允许运行符合测试标准。


　　Now we come to what constitutes “manual integration”. Figure 3 presents three options that I have selected for discussion, your laboratory SOP may have more areas depending on the work performed. The outcome you require is consistent and appropriate manual integration that is scientifically defensible. Therefore, you need to avoid situations where you have inconsistent or inappropriate integration.现在我们来谈谈什么是手动积分，图3 提出了3个观点进行讨论，你的实验室SOP根据所进行的工作可能包含更多内容。你所需要的结果是一致的，并适当的手动积分是科学合理的。因此我们需要避免一些不一致和不适当的积分。 Manual Intervention Versus Manual Integration手动干预VS手动积分


　　In Figure 3 options 1 and 2 are shown as manual intervention and option 3 as manual integration. Let me clarify my reasoning.图3中情况1和2表明手动干预，情况3是手动积分。让我来说明以下我的理由


　　Option 1: Peaks have slipped out of a window and they are not correctly identified. The automatic integration is acceptable and all that is required is to change the peak windows in the integration method and reprocess. Peak areas are not changed by this approach.情况1：峰超出了界面，他们不能被正确识别。自动积分是可以接受的，所需要的是改变积分方法的峰界面并重新处理。通过这个方法峰面积并没有改变。


　　Option 2: Parameters in the integration or processing method need to be adjusted and then applied to all injections in the run. An example could be change of the peak threshold or minimum area to reduce the impact of baseline noise. Peak areas may or may not be changed under this option but there is no manual placement of the baselines by an analyst.情况2：积分参数或处理方法需要调整，然后适用于运行中的所有进针。一个例子可能改变超过阈值角或最小面积来减少对基线噪音的影响。在这种情况下峰面积可能改变也可能不变，但是基线并没有被分析师手工处理。


　　Option 3: Manual placement of baselines by the chromatographer is required because of a late running peak or noise if undertaking an impurity analysis. Peaks areas will be changed by the reintegration.情况3：在进行杂质分析时，因为后期出现的峰(late running peak：拖尾峰?)或噪音，可能需要色谱工作人员手动处理基线。通过重新积分峰面积将会改变。

　　图3：手动干预和手动积分的建议流程图


　　Options 1 and 2 are manual intervention but the baseline placement is performed by the CDS and is not altered by a chromatographer. These are the preferred options and easier to justify scientifically. Option 3 is where everything else has failed and a chromatographer goes through individual chromatograms and repositions the baselines where appropriate. This latter point is important, it is an exercise of scientific judgement that needs to be backed up by the procedures within the integration SOP.情况1和2是手动干预，其基线位置由 CDS本身确定，没有被色谱工作人员改变。这是一个优选的情况，可以比较容易的进行科学说明。情况3在所有其他的都试过不行，色谱工作人员在适当的情况下，对色谱进行基线调整。后面这点很重要，它是一个科学判断的过程，积分SOP中需要有程序对它进行备份。


　　It’s All Plain Sailing Now?这样就好了吗?


　　Let us return to the scenario we discussed earlier at the top of the flowchart in Figure 3. If the automatic integration has failed because the peaks have slipped out of the retention windows (option 1 in the manual intervention section) do you want to trigger a laboratory investigation? Especially when the peak windows are readjusted and, after reintegration, the run now passes. The peaks are now correctly labelled and the peak areas are unchanged after the manual intervention. Although the FDA classifies this situation under “manual integration”, there is no change to the actual measurement of the peaks of interest. Should this situation be classified as manual integration? In my opinion no – this is a manual intervention but not reintegration. This is not intended to be word play but a means of trying to define exactly what the regulators want in light of zero guidance and multiple citations on the subject. Furthermore, because there is no agreed definition of the term we have a problem – would this be permitted? Would you take the Clint Eastwood approach to risk management by feeling lucky or could you justify that this is an acceptable practice?让我们回到讨论图3之前的情形，如果峰超出界面以外导致自动积分失败(情况1手动干预章节)你是否愿意发起实验室调查?特别是当峰界面被重新调整并重新积分后，运行是通过的。手动干预后现在这些峰被正确标定并且峰面积没有改变。虽然FDA定义这种情况为“手动积分”，没有改变相关峰的实际测量。这种情况是否应该被定义为手动积分?按照我的观点-这是手动干预并不是手动积分。这不是文字游戏而是在没有相关指南并多次引用的情况下，试图更加精确的定义监管者需要的是什么。此外，因为该问题没有统一的定义，我们有一个问题-这是允许的吗?你可以采用Clint Eastwood方法感觉幸运的进行风险评估或者你能证明这是一个可以接受的做法吗? Ideally the SST, standards, and QC samples should be integrated the same way consistently throughout the run; however, this may not be the case, particularly near limits of quantification or detection. Let us look at a different situation that can arise when operational efficiency is considered and different material types are analyzed in a single run. Although the method is validated for these different sample types, there can be differences in the chromatography that require different integration parameters. So how can you apply the same integration parameters across the run? The classic example is mixing APIs and stability samples where the methods have different aims; for example, determination of purity versus degradation. Perhaps the case of data integrity should win over operational efficiency and it would be better to separate different types of analytical procedure?理想情况下，SST(系统适用性试验)，标准品和QC样品应该按照统一的方法运行进行积分;然而，这可能并非如此，特别是接近定量限或检测限时。让我们来看看在考虑工作效率并且分析不同类型物料时，能够出现的不同情况。虽然方法对不同类型的样品都经过了验证，不同类型的样品其色谱图还是有所不同的，需要不同的积分参数。所以你怎么能应用相同的积分参数来运行测试?一个经典的例子是APIs和稳定性样品，这些样品的分析方法有不同的目的;例如，测定纯度VS降解物。可能数据完整性重要过工作效率，故最好分成不同的分析程序? Regardless of the content of the final SOP, chromatographers must be trained to perform manual integration using a scientifically sound, justifiable, and transparent process. We are looking at consistency of an integration technique among all trainees to ensure a consistent approach, which can be provided via a CDS by copying methods and chromatograms to a training project or directory and allowing chromatographers to integrate the same files. One outcome of the training is that trained chromatographers will think about where they place a baseline when manually integrating a peak so that it is scientifically justified and complies with the laboratory procedure.不管最终SOP的内容，色谱工作人员应该受到培训使其以科学合理并且透明的方式进行手动积分。我们需要关注的是积分技术在所有受训人员中使用同一方法的一致性，它可以通过在CDS中复制方法和色谱图来进行培训，并且色谱工作人员对同一个文件进行积分。培训的其中一个目的是受训的色谱工作人员将能够思考手动积分时基线应该画在哪里才是科学合理并符合实验室规程的。 Another outcome of the training is that all trainees must understand that unauthorized manual integration outside of the scope of the SOP is considered as data falsification. The more times that a run is reprocessed either via manual intervention or manual integration the more quality assurance and regulatory scrutiny it will attract, that is, the larger the size of the red rag. Unfortunately large red rags are not very effective at stopping regulatory bulls.培训的另外一个目的是所有受训人员必须理解在SOP范围之外的非法的手动积分是被认为是数据造假的。一个测试通过人工干预或者手动积分进行重复处理的次数越多，由此引起的质量保证和监管审查的问题越多，也就是说，红色警告的程度越大。不幸的是大量的红色警告并没有有效防止监管戒条。 As a final comment, chromatographic integration should be included in the data integrity self-inspections to ensure that the training and procedure are being complied with in operational use.最终评论，数据完整性自检应该包括色谱积分以确保在日常使用中，培训和规程得到遵循。


　　Summary总结


　　We have discussed manual integration in the context of a regulated laboratory and have found a number of problems. Regulations and guidance are lacking in GMP and the only guidance on the subject is for regulated bioanalytical laboratories, which can result in an overly bureaucratic approach. However, we lack an agreed definition of what constitutes manual integration. Not withstanding this minor problem, a suggested workflow for determining how manual intervention and manual integration can be combined in an overarching SOP on chromatographic integration to meet regulatory concerns. I hope this will stimulate a debate to define what constitutes manual integration and that regulatory agencies will provide guidance on the subject — eventually.我们已经讨论了受控试验室的手动积分和发现的一些问题。在GMP中缺乏法规和指南，这个课题上仅有的指南是生物分析室法规，并且我们没有手动积分的统一定义。避开这些小问题，通过一个建议性工作流来确定如何把手动干预和手动积分结合在一个色谱积分SOP中来满足监管的要求。我希望这能激励出一次什么是手动积分的讨论并且监管机构可以提供关于这一课题的指南。


　　1.什么情况下可以进行手动积分?


　　1)低分离度或低响应;


　　2)流动相不稳定，基线紊乱，保留时间变化小;


　　3)色谱柱性能：运行过程中，出现异常峰;


　　4)软件积分的局限性，如由于软件参数阈值的设置导致峰未积分，峰起点和终点间出现肩峰和异常的基线漂移，软件错误(峰未积分和错误识别峰);


　　5)由于样品母体的干扰导致的复杂色谱图：裂峰、目标杂质的同时洗脱/肩峰、基线噪音、负峰、基线上升或下降(由于某些梯度程序)、峰的严重拖尾、烃类的存在导致自动积分不正确。


　　2、关于手动积分的检查不合格项


　　在 2014年，FDA对欧洲某实验室的检查中，483表格中有一个观察项这样表述：没有描述如何进行手动积分的操作规程(Noprocedure exists describing how to perform manual integration)。在2007年8月，FDA给Leiner Health Products公司的警告信中，有一条缺陷项这样表述：检查员记录了很多“数据捏造(manipulation of data)，却没有解释捏造原因”。这个捏造包括：改变色谱积分参数、重新标记峰，这样之前本应该积分的峰就不再积分，不计入杂质的计算。这说明了FDA 是接受手动积分的，但应该有操作规程控制手动积分的条件、权限、注意事项等，不能背离科学地通过手动积分使检验结果合格。


　　备注：在FDA的现场检查中，例如发现预进样、重复检验的情况，检查员的观点是：如果企业证明是科学的、必要的，就应该写入操作规程。这也意味着，需要经过充分验证、科学评估。


　　3、手动积分的用处


　　理想条件下，在通过系统适用性试验后，标准品、样品的色谱应一直以相同的方法进行积分。但是实际情况并非如此，尤其是接近定量限、检测限的情况下，有些峰自动积不上，需要手动积分。手动积分虽然会受主观因素的影响，在科学分析色谱结果时也是需要的。举两个例子说明：


　　例1，当出现异常峰，分离度不好的峰时，可以手动强制分割。


　　例2，研发阶段，API、稳定性样品的纯度、降解产物的检验方法色谱运行条件相同，但应考虑到检测分析的重点不同，可能需要不同的积分参数。在这种情况下，最好建立不同的分析方法、程序。


　　4、关于手动积分的指南


　　关于手动积分，并没有指南给出明确的定义。笔者只查到有两篇生物样本分析的方法验证指南规定了再积分(Reintegration)的注意事项。


　　FDA 的2001年定稿指南 Bioanalytical Method Validation，及相应的2013年草案指南也有类似的要求。 生物样本分析的方法验证2013草案指南，第III.C节“样本数据再积分”规定：SOP应该确定再积分的标准，和怎样进行再积分;应清楚描述、并记录再积分的理由;应报告原始和再积分数据。第VII.D节还规定：再积分数据应有管理人员授权再积分。


　　EMA 的2011年指南 Guideline on bioanalytical methodvalidation，第5.5节规定：应该有SOP描述色谱的积分和再积分。应在分析报告中讨论偏离这个SOP的任何偏差。应该记录色谱积分、再积分的 积分参数、初始积分数据、最终积分数据，并在要求时立即可得。


　　5、没有公认的手动积分定义


　　目前没有关于手动积分的公认的定义，Hill的一篇文章，讨论了手动再积分的范围和术语，并总结认为，建立一致的定义是有必要的。如果没有手动积分的定义，企业怎么建立积分的SOP呢?


　　在2015年11月，美国费城的ISPE年会上，企业人员Bob McDowall和Mark Newton建议区分手动干预(Manual Intervention)，和手动积分(Manual Integration),并提出：


　　手动干预 ：没有手动改变基线，例如：1)对峰重命名，或调整积分窗口;2)改变积分参数;


　　手动积分 ：分析员手动重新划基线。


　　举两个例子说明：


　　例1，在色谱峰的保留时间不在预期的时间窗口内，需要手动干预时间窗口，而峰面积不会改变;需要调查保留时间偏离的原因。可能FDA会把这个干预也归为手动积分;但是Bob认为不必归类为手动积分。所以希望监管机构给出明确定义的指南。


　　例2，有IPEM学员单位提议：手动干预、手动积分这两种方式在有些情况下可替代使用的。峰无法识别(或不能正确识别)时可能通过手动积分(重画基线)或调整积分参数解决;峰积分不好时可通过手动积分(重画基线)或调整积分参数解决。


　　6、手动积分使用的注意事项


　　通常，在得到自动积分色谱图后，分析保留时间、峰型是否如预期，峰是否被正确识别，基线是否满足要求，或者其它标准。只有在色谱图不可接受时，才可考虑是否可以进行手动积分，或者进行实验室调查。


　　有两种情况，是禁止进行手动积分的：


　　在自动积分后，对称的峰有可接受的基线;


　　增强峰、或削减峰的目的是为了满足系统适用性接受标准，或满足放行质量标准。


　　总之，企业应建立科学、合理、透明的自动积分操作规程，并满足数据完整性的要求。在SOP之外进行的手动积分可能被视为数据造假。


　　Data Processing and Peak Integration


　　数据处理和峰的积分


　　Chromatographic software uses dynamic peak detection algorithms and automatic peak detection algorithms. Integrating the chromatograms using software to apply the integration events, such as peak width, threshold, height, and area, etc., and then visually observing the peak integration for its correctness is generally recommended. The inherent or analytical variations and combination of various and drifting baselines in which the auto-integration will either underreport or overintegrate the peak areas.


　　色谱软件使用动态寻峰算法和自动寻峰算法。通常建议先用软件积分色谱来处理像峰宽、阈值、峰高及峰面积等需要积分的情形，再人工确定积分的正确性。因为一些固有的或分析变化或两者皆有以及基线漂移的原因，自动积分将会使峰面积偏大或者偏小。


　　Once acquired, the electronic raw data of the measurements (measured values and metadata) are stored and available for processing. Some acquired electronic raw data already represent usable results (e.g., weight, temperature, and humidity). Other acquired electronic raw data, such as intensity values correlated with time or wavelength and generated by chromatography or spectroscopy, require further processing to obtain usable results (e.g., retention times, peak areas, and amounts). These processes, such as integration and calibration, are defined by processing parameters or calibration factors and affect only the resultant data after processing, but not the acquired electronic raw data. In contrast to the acquired electronic raw data, the processing parameters, such as integration events and calibration curves, may be changed during data evaluation. The changed processing parameters, methods, and processed data should be identified by versioning (i.e., number of times reprocessed) either on the results or from the audit trail data (25).


　　实验的电子原始数据(测试结果和元数据)一经(色谱软件)获取，将会被(软件)存储并可用来进行处理。那些诸如重量、温度、湿度等电子原始数据在被获取后即可直接作为结果;而其他由色谱或光谱产生的与时间或波长相关的电子原始数据，则需要经过进一步处理才能够得到如保留时间、峰面积以及物质的量等可以被使用的结果。像积分和校正这些仅由处理参数或校准因子所定义的处理过程，它们只影响处理后的最终结果，而对电子原始数据没有影响。与获得的(确定的)电子原始数据相反，在数据评估过程中，积分事件和校正曲线等处理参数可能发生改变。改变了的处理参数，方法以及处理后的数据应该标示版本控制的方法标记在结果或在审计追踪中。


　　Integration events should be defined through standard algorithms, and metadata and should be associated with respective raw data files. In such cases, the analyst adjusts the integration events to obtain proper peak integration. As chromatography is a comparative technique, the consistent integration events should be applied for the entire set of chromatograms, as much as possible, or published along with the chromatograms. Figure 6.3.7-1 represents an overlaid chromatogram of the standard and a sample (of the same concentration) integrated with the same integration events. Integrating small peaks, closer peaks, negative peaks, drifting peaks, and peak-to-valley requires time and skill. Integrating peaks manually is not recommended and should be avoided to the extent possible.



　　积分事件应该通过标准算法和元数据进行定义，且处理后的结果应与各自的原始文档进行关联。只有这样，分析师才可以通过调整积分事件从而获得适当的峰面积积分。因为色谱是一种比对技术，所以应尽可能的将同一积分事件应用于整个色谱图集或和整个色谱图一起发布。图 6.3.7-1表示标准品和某浓度下的样品通过同样的积分事件进行积分，并一同呈现在同一色谱图中。对小峰、接近峰、负峰、漂移峰以及峰谷进行积分需要时间和技巧。不推荐手动积分，且应尽可能避免进行手动积分。

　　QC laboratories should have procedures in place that require authorization to perform manual integration and for procedures to track such events to avoid unnoticed or unevaluated cases that may affect the accuracy of the results. PDA defines manual integration as a process used by a person to manually integrate the peak height or area by modifying the baseline of the chromatogram with use of chromatographic software. The conditions and circumstances when manual integration would be allowed should be predefined (e.g., complexity of the sample matrix). Generally, a good chromatographic data system would be able to render consistent and reliable baselines for an overwhelming majority of injections within a chromatographic run. When consistently bad chromatographic peaks and baseline issues are encountered, having good documentation should not be the only way to address the issue. In this circumstance, the goal should be to improve the system and ensure that the data generated is reliable and consistent.


　　QC实验室应建立需要授权才能进行手动积分的程序并追踪此类事件(注：手动积分)的程序，避免因为人员忽视或者未评估而影响结果的准确性。PDA将手动积分定义为：由人员使用色谱软件修饰色谱图的基线来人为对峰高或者峰面积进行积分的过程。应当事先定义允许进行手动积分的条件和情形(如：复杂的样品基质)。通常，一个良好的CDS系统应该能够在色谱运行中为绝大多数进针提供连续且可靠的基线。当色谱峰连续比较差且发生基线发生问题时，将问题完整的记录下来并不是唯一的解决途径。在这种情况下，应该以改善系统并确保产生的数据真实可信为目的。


　　The Quality Unit should define standard protocols for processing data to include the following, which may be instrument or application-specific:


　　质量部门应定义处理数据的标准方案，可以是针对仪器或特定应用。方案应包含以下内容：


　　Reprocessing peaks


　　峰重新处理


　　Applyingin strument/application-specific integration events/algorithms


　　使用仪器/特定软件的积分事件/算法


　　Fully integrating peaks


　　峰完全积分


　　Inhibiting ordisregarding any peaks in the test chromatogram (e.g., blank peaks, placebo peaks, solvent peaks) without scientific justification; examples where justification is needed include counter ion and reagent interactions with a sample


　　禁止在没有科学论证的情况下，忽略测试色谱图中的任何峰值(如：空白峰，安慰剂峰，溶剂峰)。如样品与反离子或试剂相互反应需要科学论证。


　　Applying thesame integration events for all samples in the sample set or sequence andjustifying any change in integration events.


　　对处在同一样品集或序列的样品采用同样的积分事件，并说明积分事件的任何改动。


　　Peak integration is the process used by a chromatographic system to determine the peak height and width and obtain the quantitation of the peak of interest. Certain USP monograph methods specify inhibiting integration at specified zones in the chromatographic run. USP <621> states that peaks can be disregarded by setting the thresholds in the integration to at least half of those below reporting threshold(32). Thereby, utilizing the built-in capability of the chromatographic data acquisition software to inhibit integration of peaks from solvent, mobile phase, placebo, and counter ions in impurity analysis in a common industry practice. Firms should scientifically evaluate and judiciously determine whether to use the inhibit integration functionality. Peaks in the chromatographic analysis may be excluded in the event of a known abnormality. Unknown peaks should be integrated and investigated according to the firm’s quality procedures.


　　峰(自动)积分是色谱系统用来确定目标峰峰高和峰宽以及定量的过程。某些USP专论方法指出在色谱运行时禁止(系统)对特定区域进行积分。USP<621>指出只有在通过对积分中的阈值进行设置(低于报告阈值一半以上)(32)，才可以将那些低于报告阈值一半以上的峰忽略。因此，行业的普遍做法是，在杂质分析时利用色谱数据采集软件(CDAS)来防止对溶剂、流动相、安慰剂和反离子产生的峰进行积分。公司应科学合理的评估和决定是否使用抑制积分功能。在异常已知的情况下，可以将谱图分析中的一些峰排除。而未知峰应根据公司的质量程序进行积分和调查。


　　Manual integration is the process used by the analyst to integrate the peak height or area by modifying the baseline of the chromatograph using software(49). Manual integrations may be required for R&D and biological labs. Though in QC labs, manual interagtion may be necessary or acceptable only under special circumstances (e.g., complex chromatography due to sample matrix interferences, poor resolution, co-elution of peak of interest, problem with the baseline, or software with limited capabilities); however, manual integration is not generally accepeable for assay. Manual integration should not be left to an analyst’s discretion; it should be performed only according to an approved procedure, with documented approval from a supervisor and results appropriately documented. A mature quality system will review these instances as part of continual improvement of methods and equipment. Modern chromatographic software identifies and displays manually integrate peaks. Figure 6.3.7-2 represents a model chromatogram with manually intergrate peaks and software integrated peaks.


　　手动积分是分析师通过使用软件对色谱图基线进行修改从而对峰高或峰面积进行积分的过程(49)。研发和生物实验室可能需要(对色谱图)手动积分。尽管对于QC实验室，手动积分可能仅在特殊情况(如：由于样品基质的干扰所引起的复杂图谱;分辨率差;目标峰的共洗脱，基线问题以及系统能力有限)下是必需的或可接受的;但手动积分还是不适合应用于化验。手动积分只有在得到主管书面批准且结果正确记录后，根据批准程序才能进行，而不应该由分析师自行决定。一个成熟的质量体系应审查这些实例，作为方法和设备改进的一部分。现在的色谱软件已可以识别和标示出手动积分峰。Figure 6.3-7.2 是一个模型色谱图，它同时展现了手动积分峰和软件积分峰。

　　Printed chromatographic should be presented in visible scale as per the respective analysis(peak top visible for assay or single analysis, peak base clearly visible for purity analysis). After integration, results may be publish or electronically stored. If results are reprocessed, permission from supervisor is required. Those types of events may be noted in a log (paper of electronic) for quick reference.


　　打印出的色谱图应以可见比例形式呈现各个分析项需要的内容，如对于含量和单个分析项，峰顶应可见;对于纯度分析，峰基应可见。在积分后，结果应能够输出或以电子形式存储。如果结果被重新处理了，那么应得到主管的许可。可以将这些时间记录在日志(纸质或电子形式)中，以供快速参阅。


　　Figure 6.3.7-3 illustrates a chromatogram in proper scale and visibility to determine the proper integration. Common practice across the industry is to present a visible and clear baseline for multicomponent analysis and the entire peak for single-peak analysis.


　　Figure 6.3.7-3以一个有着适当比例和可见的色图谱来说明适当的积分。行业内通常的做法是为多组分分析提供清晰可见的基线，而为单峰分析提供整个色谱峰。

　　Figure 6.3.7-4a represents to the chromatogram printed in large scale, where peak integration is not visible. Figure 6.3.7-4b represents the same chromatogram with printed proper scaling, where peak trimming and missing peak integrations are visible, revealing possible intentional data manipulation.


　　Figure 6.3.7-4a是一张以最大比例打印的色谱图，其中峰值积分并不可见。Figure 6.3.7-4b是同样的色谱图以适当的比例打印，其中可以看到峰值修正和缺失峰的积分，揭示了可能的有意的数据操作。

　　Assay analysis, dissolution, and content uniformity involve a single analyte/peak analysis where the content will be calculated against a known standard. Gaussian peaks, with allowed system suitability characteristics of accuracy and precision, are expected in these types of analysis. Normal Gaussian peaks should be integrated exactly where the peak starts and finishes. The peak integragtion represented in these types of analysis is illustrated in Figures 6.3.7-5a-f.


　　含量，溶出度以及含量均一性包含了对单一分析物/峰按照已知标准进行计算的分析项目。在这些类型的分析项目中，都期望其具有准确和精确的系统适应性特征的高斯峰。正常的高斯峰应该确切地从色谱峰起始位置开始积分到截止为止结束积分。Figure 6.3.7-5a-f 展现了这些类型测试代表性的峰积分。

　　Figure 6.3.7-5a is the unprocessed signal of a single component analysis. Figure 6.3.7-5b represents raw data integrated with proper integration events and displayed in visible scaling. Figure 6.3.7-5c represents the peak integrated by trimming to yield low area. (Associated risk: Reporting fewer peak areas leads to less assay or dissolution or lower content values.) Figures 6.3.7-5d-f represent the raw data integrated with improper integration events, which presents more area. (Associated risk: Reporting more peak areas leads to more assay or dissolution or higher content values.)


　　Figure 6.3.7-5a是单组分分析的未处理的信号峰。Figure 6.3.7-5b表示的是对原始数据以适当的积分事件进行积分并以可见的比例呈现。Figures 6.3.7-5c 表示的是通过修整积分而产生了低面积的峰。(相关风险：报告峰面积的减少会导致含量或溶解度的降低或含量值的减少)Figures 6.3.7-5d-f 表示的是对原始数据用不适当的积分事件进行积分会表达更多的面积。(相关风险：报告峰面积的增加会导致含量或溶解度的增加，或含量值的增加)


　　Data manipulation of chromatograms by manually manipulating the integrated peaks is one factor the FDA has cited in Warning Letters(50-53). In these illustrations, all the integrations presented are made through chromatographic software by adjusting integration events. Chromatographic software will indicate which peaks are maually integrated. Laboratory management should have appropriate controls in place for detecting the data compromises made by applying the wrong integration events.


　　手动操作积分峰对色谱图进行数据处理是FDA在警告信中引用的一个因素。在这些图示中，所有的积分都是通过调整色谱软件的积分事件来完成的。色谱软件会标示出哪些峰是手动积分的。实验室应该有适当的控制措施以检测应用了错误的积分事件而造成的数据损毁。


　　Figures 6.3.7-6a-c and Figures 6.7.7-7a-c show integration errors that occur for multicomponent analysis (peak groups) , such as related substances analysis/chromatographic purity analysis. In both cases,fewer peak areas will reported.


　　Figure 6.7.7-6a-c 和Figure 6.7.7-7a-c展示的是在如成分分析/色谱纯度分析这种多组分分析(峰组)中的发生的积分错误。这两种情况下，报告的峰面积都会减少。

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