手动积分与自动积分如何定义？

没考虑过手动积分的问题，学习了

色谱积分原理和手动积分的规则
原创： GMP办公室翻译组 GMP办公室 2016-10-02

Questions of Quality: Where Can I Draw The Line?质量问题：我能在哪里划这条线？A question that keeps raising its head when working in a regulated laboratory is can chromatographers integrate peaks manually? If they can, when can they do it? Also if they can manually integrate, when should they not do it?一个问题正在持续发酵并且占据了头条：在受控实验室何时能手动去对色谱峰积分？如果他们能这样做，何时可以做呢？同样如果他们能够手动积分，何时不能这样做呢？

One of the red rags to a regulatory bull is the issue of manual integration of chromatograms. If seen during an inspection, you can almost see the wheels in the inspector’s brain turn in mechanical precision as they question: are they testing into compliance? In a 2014 FDA inspection of a laboratory in Europe, one FDA 483 observation stated:色谱手动积分问题就像是斗牛中的一块红布。如果在检查期间被发现，你几乎能够看到检查官的脑中条件反射地联想到一个问题：他们的检验是否合规？在2014年FDA检查一个欧洲试验室时，一个FDA483的缺陷项如下： No procedure exists describing how to perform manual integration.没有关于怎样进行手动积分的规程。 A more serious non-compliance occurred in the FDA warning letter to Leiner Health Products:在对Leiner Health Products 的FDA警告信中提到一个更加严重的不符合项： In addition, our investigators documented many instances with extensive manipulation of data with no explanation regarding why the manipulation was conducted. This manipulation would include changing integration parameters or relabelling peaks such that previously resolved peaks would not be integrated and included in the calculation for impurities.

此外，我们调查人员记录了许多关于修改数据的案例，他们都没有解释为什么修改。这些修改包括改变积分参数或者重新标定峰以至于原来峰的分离度不能进行积分和杂质计算。

There have been many other cases that I summarized in a recent Questions of Quality column on the role of chromatography data systems (CDS) in data falsification. What regulatory guidance is there to help us? We need to consider some basics of integrating chromatographic peaks before we can look at manual integration and the regulatory guidance surrounding it. The reason for this is simple: if integration parameters are set correctly and the chromatography is acceptable then there should be no need to reintegrate manually in many cases. However, we also need to acknowledge that chromatographic systems are dynamic by their nature and separations can change during a run, so getting the right overall integration can be a balancing act.还有一些其他情况，我近期总结了关于一个色谱数据系统（CDS）在数据造假中的作用的质量问题专栏。在这里有什么法规指南能够帮助我们？我们在关注相关积分手册和法规指南之前需要先了解一些色谱积分的基础问题。这样做的理由很简单：如果正确设定积分参数和可接受的色谱图，那么一些情况就没有必要进行手动重新积分。但是，我们需要承认在检测运行期间色谱系统的特性是动态的和分离是可变的，因此正确全面的设定积分能起到平衡的作用。 The regulators are wrong in their requirement for a procedure on manual integration. What is required instead is a standard operating procedure (SOP) for chromatographic integration, of which manual integration is an important sub-set. Although the focus of this column is manual integration please do not lose sight of the bigger picture. As such, we will not be discussing the various calibration methods that could be applied to standards to quantify analytes in samples nor will we be looking at analogue to digital conversion because the latter has been covered in an earlier Questions of Quality column . Furthermore we will not be considering the use of system suitability tests (SSTs), again because this column has already discussed them . The paper by Hill et al. on manual reintegration in bioanalysis is also a highly recommended publication to read on the subject.监管者对手动积分规程的要求是错误的。这些要求对色谱积分的标准操作规程（SOP），而不是手动积分的要求，手动积分是另外一个重要分支。虽然专栏的焦点是手动积分，但是我们要比较全面的来看待问题。因此我们将不讨论适用于标定样品定量分析使用的各种校验方法，也不去关注类似的数据转化因为这些已经在之前的质量问题专栏中提到过了。此外我们也将不考虑系统适用性（SSTs）的使用，因为已经再其他期的专栏中讨论过了.本文强烈推荐去阅读Hill et al关于生物分析的手动重新积分的课题. Back to Integration Basics

色谱积分基础

Before we can discuss how to control and manage manual integration it is important to understand the basics of chromatographic integration itself. The best book on the subject of integration is by Norman Dyson and, although the second edition was published in 1998, it is still applicable and highly recommended if you want to understand chromatographic integration. The key integration parameters are shown in Table 1 and the main ones are discussed below. Note that some CDS suppliers may call these parameters a different name but the functionality is essentially the same.之前我们讨论如何控制和管理手动积分，这对于色谱积分本身基础的理解是十分重要的。对于积分这个课题最好的书是Norman Dyson 的著作，在1998年已经发行了第二版，它仍旧使用。如果你想了解色谱积分强烈推荐这本书。关键积分参数在表1中列出，并且一些重要的部分在下面进行了讨论。值得注意的是不同的CDS供应商对他们的参数有不同名称，但是功能的本质是相同的。

图1：峰的关键参数

对于峰值采集和测量CDS积分的关键参数

Chromatographic integration begins when the CDS samples the detector output using an analogue to digital converter (A/D). An integration method in the CDS determines the frequency of the detector data collection and analysis run time. Dependent on the CDS, either an integration method or a processing method will be automatically applied to the file of data slices to calculate the peak areas or heights. The processing method will contain the identities of the peaks of interest and their expected elution time windows. These data files and their interpretation constitute part of the raw data and primary record for the analysis. We will discuss the definition of primary record in the next Questions of Quality column in light of the two MHRA data integrity guidance documents issued this year .

当检测器用一个类似于数字转换器（A/D）的装置开始输出信号时色谱积分开始。在CDS中积分的方法确定了检测器收集频率和分析运行时间。依靠CDS，积分方法或处理方法将自动应用于峰面积或高度的计算积分。处理方法包括对目标峰的鉴别和期望在界面中的洗脱时间。这些数据文件和解释组成了分析的原始数据和最初记录。我们将讨论在下一个质量问题专栏中讨论最初记录的定义，根据MHRA发布的数据完整性指南。

The two most important parameters for integration are sampling rate and peak threshold. In combination they determine the slices for peak measurement but also suppress noise to allow better peak measurement .

采样率和超过阈值角是积分的两个最重要参数。结合他们两个决定了峰测量，但抑制噪声能够使峰得到更好的测量。

Peak width or sampling rate setting governs how often the detector output is sampled, which directly impacts the accuracy of peak area measurement. If the sampling rate is too slow it could result in the integration missing a small fast eluting peak or a valley between two peaks. Conversely, too fast a data acquisition rate can be managed by data bunching in the CDS software. Typically you should sample a peak at least 20-30 times across its width so that it can be integrated accurately; for example, conventional high performance liquid chromatography (HPLC) typically requires a 0.5-1 Hz sampling rate, faster capillary gas chromatography (GC) has a sampling rate in the range 5-20 Hz, and ultrahigh-pressure liquid chromatography (UHPLC) needs a sampling rate between 20-50 Hz. Most A/D units in CDS are rated at up to a 100 Hz sampling rate.

切线峰宽（peak width）和采样率的设定控制检测器采样的输出频率，并对峰面积测量的准确度有直接影响。如果采样率太慢，可能导致积分不到小的洗脱峰和两峰之间的低谷。相反，太快的采样频率会被CDS软件中的数据聚束所管制。一般情况下，至少20-30次采样才能准确的积分。例如，传统HPLC一般要求0.5-1HZ的采样率，快速毛细管气相GC的采样率范围5-20HZ，UHPLC的采样率范围是20-50HZ.大多数在CDS中的A/D中的采样率上限是100HZ.

• 
  
  Peak threshold is the setting that discriminates the start and end of a peak from baseline noise. It is based on the rate of change when the detector signal rises above a preset value in the integration method for peak start or falls in the case of peak end. This needs to be set carefully because if the baseline is noisy then noise is detected as peaks; if set too large the integration will miss small peaks because the threshold is not triggered. The same process occurs at peak end but if the threshold is low, too much of the peak tailing is counted. Conversely if the threshold is set too high the peak finishes early, which under-estimates the area of the peak.
  超过阈值角（Peak threshold）设定用于辨别一个峰在基线噪音上开始和结束。它根据当检测器发出大于以上预定值的信号时发生频率的改变在积分方法中由于峰的开始或峰底部下滑的情况。他需要小心设定，如果不小心基线被检测成噪音，然后噪音被检测成峰；如果设定太大积分将可能错过小峰，因为阈值没有响应到。相同的程序也可能发生在峰底部，如果阈值设定太低，就有太多的峰尾被记录。相反如果阈值设定过高，峰会很早结束，峰面积会被低估。
  

• 
  
  Once the peak has been detected the next step is to determine when it ends. Typically this is when the signal returns to where the baseline was before the peak started but if there are several peaks eluting or the baseline is rising then the signal will not return to the baseline origin. Therefore baseline drift tolerance determines how far the baseline can drift from the original position. This is in contrast to the peak threshold, which determines how fast it can drift away (8).
  一旦峰被检测到下一个步骤就是确定它何时结束。一般情况下信号会返回原来峰开始时的基线位置，但是如果有几个峰洗脱或者基线上升，信号将不能返回到原来的基线位置。因此基线漂移公差确定了基线能够漂移原来位置有多远。这是以超过阈值角为参照，它确定基线能漂移多远。
  

From this and alongside a more detailed discussion in Dyson’s book (8) we can develop three basic rules of chromatographic integration:

在Dyson’s book 中有详细的讨论，我们能够开发出色谱积分的三个基本原则：

• 
  
  Rule 1: Do NOT use default integration parameters. Ensure that each set of integration parameters is tailored to an individual analytical procedure and do not use a one-size-fits-all approach. For Beer‑Lambert’s law to hold, the samples and standards must be consistently integrated, otherwise the fundamental comparison of absorbance versus concentration cannot be performed.
  原则1：不使用系统默认的积分参数。确定每个积分参数被设定为适应单独的分析程序，不能用一个放之四海而皆准的方法。对于Beer‑Lambert定律，样品和标准品必须连续积分，否则吸收度与浓度的对比不能执行。
  

• 
  
  Rule 2: The function of a CDS is not to compensate for your poor method development or separation. There is a belief in many laboratories that a CDS can be used to salvage an analytical run but this is not the case. Robust and validated methods should reduce or eliminate this issue.
  原则2：CDS的功能不能弥补你方法的缺点。许多实验室中有一个信条，CDS能用于弥补分析方法的缺陷，其实事实并非如此。健全和经过验证的方法能减少或消除这一问题。
  

• 
  
  Rule 3: Understand what is happening in the CDS. Just because you get a number from a CDS does not mean you have to believe the result. Use your eyes to look and your brain to think. For example, look at the integration codes (BB or BV): are these the ones expected? Are baselines positioned where they should be and are they as expected? Are retention times and peak shapes consistent throughout the run?
  原则3：理解CDS发生了什么的事情。因为从CDS中得到一些不明信息，但是你必须相信他的结果。所以只有用眼睛去看观察用脑子去思考。例如，观察积分代码（BB 或 BV)：这是预期的吗？基线应该在什么位置，希望他们在什么位置？是否保留时间和峰的形状是否始终如一在运行中？
  
  

To complete this overview of CDS integration, please note what Dyson says: improving the chromatography must always take precedence over setting up the CDS (8):

读完CDS积分的概论，请注意Dyson说的话：改善色谱方法优先于优化CDS

If a chromatographer finds it necessary to tweak the parameters continuously in order to achieve consistent measurement of standard samples, it is a clear indication that more work is needed to bring the instrument and analysis under control.

如果一个色谱方法需要不断的调整来达到测量标准品的目的，这就说明，需要更多的工作来使仪器分析受控。
In essence the likelihood of a Leiner‑style non-compliance citation is looming. This is because excessive use of manual integration to compensate for poor method development or chromatographic separation increases risk to data integrity (accuracy), and increases time to generate, review, and release results.

其实Leiner‑style不合规的可能性正在逼近。这是因为过多的手动积分来弥补方法开发的缺点或色谱分离将增加数据完整性（精度）的风险，同时增加了生成、审核和结果放行的时间。
How Can Manual Integration Result in Falsification?
手动积分如何导致了造假？

Using manual integration to falsify chromatographic data can arise in a number of ways, but the two main ways are:

用手动积分来进行数据造假的情况正在增加，但是主要有两种途径：

• 
  
  Peak Shaving — Manually placing the baselines to reduce the peak area on integration to enhance the analyte amount in a sample (only the standards are shaved) or reduce the amount of analyte reported (by shaving the sample and not the standards). In Figure 2 the first eluting peak has had the baseline manually adjusted to reduce the total peak area.
  峰值消减-手动设置基线来减少峰面积在积分中增加样品的分析数值（近标准品面积被消减）或者减少分析报告的数值（通过消减样品的峰面积但不减少标准品的面积）。在图2中第一个洗脱峰被进行了手动调整同减少总的峰面积。
  
• 
  
  Peak Enhancing — Adjusting the baselines for integration to increase the area of a peak. The enhancement of sample areas over the standards can increase the calculated amount in the sample. If reduction is required, the enhancement of the standards only is performed. Figure 2, line 3 shows how the minor or second eluting peak in the chromatogram can be enhanced.
  峰面积增加-在积分过程中调整基线增加峰的面积。增加样品的峰面积多于标准品的面积，从而增加样品的计算数值。如果需要减少，仅增加标准品的峰面积。图2中，line3显示了怎样增加色谱图中的第二个洗脱峰。
  
  

Figure 2 shows the exaggerated enhancement and shaving of peaks but sometimes all that is required is a small change to bring a non‑conforming batch into compliance with a specification. Hence the need to trend analytical results as required under the new revision to EU GMP for Quality Control laboratories (12) to highlight out of expectation (OOE) and out of trend (OOT) results, as well as those that are out of specification (OOS). Results out of trend or expectation may identify the actions of an individual analyst, which may warrant closer inspection.图2中显示夸大的增加和消减峰面积，所有这些都是让结果发生轻微改变从而使不符合变成符合标准。因此需要按照新版EU GMP对QC控制要求，对分析结果进行趋势分析，加强OOE,OOT和OOS结果的控制，OOT或OOE的结果可能有别于其他单个分析活动，但是可能需要更仔细的检查。

图2： 峰面积增加和消减的例子
In addition, inspectors and auditors will look for a number of factors to determine if there is unauthorized manual manipulation of a peak:

另外，检查官和审计人员应该关注一些非法手动积分的相关因素：

• 
  
  Discovery of manual integration that is not traceable or retrievable. A chromatographer should be able to demonstrate that same results can be obtained if data files from a run are reprocessed — a red flag should be raised if this cannot be done quickly. An inspector looking to see if the CDS audit trail has been turned off temporarily to conduct falsification could also accompany this
  发现一些手动积分没有追踪和不可读。色谱工作人员应该能演示获取相同的结果如果数据文件来源于一个重新处理的序列-如果不能获取，一个警告就应该出现。检查官发现CDS的审计追踪系统已经临时关闭，就可能伴随造假了。
  
  
• 
  
  Electronic data are not available. A focus on paper because the raw data and the electronic records have been deleted or not saved means that data cannot be reprocessed and the result confirmed. The question of falsification is raised and the laboratory is on the slippery slope to compliance hell.
  电子数据不可用。因为原始数据和电子记录已经被删除或不能被重新加工而没有留存，这个结果一旦证实。造假的问题就出现了，并且实验室符合度随之下滑至谷底。
  

• 
  
  Integration parameters are different for standards and the unknown samples of the same run or between replicate injections of the same unknown sample in an analytical batch.
  相同运行中标准品和未知样品设定不同的积分参数或者在批分析过程中重复进样未知批的相同样品。
  

• 
  
  Evidence that the audit trail in the system has been turned off and then on again a few minutes later: what changes have been performed that are not recorded?
  在系统中关掉审计追踪几分钟后再次开启提示：改变了什么不被记录的意图？
  
  

GMP Regulations and Regulatory Guidance

GMP法规和监管指南
Although there is great interest in data integrity in laboratories working under the Good Manufacturing Practice (GMP) regulations, there is a paucity of either explicit regulation or regulatory guidance to help chromatographers.

虽然GMP管理中有许多实验室数据完整性方面的法规，但是缺乏对色谱分析的明确规定或监督指南。
The only GMP regulations applicable are the requirements for scientifically sound analytical procedures in 21 CFR 160(b) and for complete data under 21 CFR 211.194(a) . The latter topic was discussed in two earlier QOQ columns . In addition, there is data integrity audit under objective 3 in the FDA’s Compliance Policy Guide 7346.832 for pre-approval inspections . Both the European Pharmacopoeia (chapter 2.2.46)and the United States Pharmacopoeia <621> discuss criteria for good chromatography; there are no criteria for chromatographic integration or a similar discussion on data integrity. However, in the interest of scientific soundness: can you justify and defend your actions on the basis of good chromatographic science?

能使用的法规仅有科学合理的分析方法21 CFR 160(b)和完整的数据21 CFR 211.194(a) ，后者在早期的QOQ专栏中讨论过。另外，在objective 3 in the FDA’s Compliance Policy Guide 7346.832 for pre-approval inspections 中有数据完整性的审计。在the European Pharmacopoeia (chapter 2.2.46) and the United States Pharmacopoeia <621> 中说明了良好色谱分析的标准。没有相关色谱积分的标准或相似的数据完整性讨论。但是为了科学可靠的目的：您能证明和说明你的活动是基于科学的色谱分析吗？
In my view, there is a need for regulatory agencies to issue guidance on the subject of integration with a focus on manual integration for GMP. As they typically move at glacial speeds, the likelihood of this occurring before the next ice age is probably minimal. However, there is an area where regulators have been active on the subject of manual reintegration and this is in bioanalysis of samples from non-clinical and clinical studies for the registration of drugs.

我认为，这需要法规监管部门发布一个关于GMP手动积分主题的指南。但这是特别缓慢的。说不定可能会到下一个冰河世纪。但是有一个地区已经对手动积分做了行动，这就是药品注册时临床和非临床研究的样品生物分析。
Bioanalytical Regulatory Guidance for Reintegration

生物分析法规指南中的重新积分
The FDA created guidance for bioanalytical method validation following an AAPA-FDA conference in Washington in 1990, where I was involved as a co-chair, and the outcome from that meeting was a scientific paper on the subject . After a follow-up conference in 1999, the FDA issued Guidance for Industry on Bioanalytical Method Validation . In the section dealing with routine analysis of samples there is mention of sample data reintegration:

1990年FDA对生物分析方法验证建立了指南在Washington的AAPA-FDA会议中，我作为联合组长参与了这次会议，这次会议的结果见scientific paper on the subject 。后续的会议在1999年召开，FDA发布了Guidance for Industry on Bioanalytical Method Validation ，在这个部分中关于处理日常样品检验的重新积分观点如下：

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  An SOP or guideline for sample data reintegration should be established.
  应该建立一份关于样品数据重新积分的SOP或指南。
  
• 
  This SOP or guideline should explain the reasons for reintegration and how the reintegration is to be performed. The rationale for reintegration should be clearly described and documented.
  在SOP或指南中应该说明重新积分的原因，并说明如何进行了重新积分。重新积分的原理应该被清楚的描述和记录。
  
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  The original and the reintegration data should be reported.
  初始和重新积分的数据都应该被报告
  

Later in the document, in the section on reports for routine studies, there is another section on Documentation for Reintegrated Data, which, in part, requires:

在后来的文档中，在日常研究的报告章节，有另外一个文档Reintegrated Data ，要求如下：

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  The method used for reintegration.
  重新积分使用的方法
  
  
• 
  The reason for the reintegration.
  重新积分的理由
  
  
• 
  The requestor of the reintegration and the manager authorizing reintegration.
  重新积分的申请者和管理者授权后才能进行重新积分。
  
• 
  Reintegration of a clinical or preclinical sample should be performed only under a predefined SOP.
  临床和临床前样品的重新积分只能在预先指定的SOP下进行操作。
  
  

In 2013, the FDA issued a draft revision of this Guidance for Industry where sample data integration was updated (please remember that the guidance is still a draft), but the key requirements are essentially unchanged. There is a rearrangement of the order listed above with the additional need to retain audit trail information from the CDS .

在2013年，FDA发布了对工业生产的指南修改起草版本，样品数据积分进行了更新（但是请注意现在这个指南依然是起草版本），但是关键的要求没有进行本质的改变。是对以上文件的重新整理文件，除了增加了审计追踪的在CDS中的保留部分。

At last we are getting somewhere! We now have some guidance albeit in the good laboratory practice (GLP) arena. But wait! There is more! Not to be outdone by their American cousins, the EMA (European Medicines Agency) produced their own guidance document on Bioanalytical Method Validation (22) in 2011. Section 5.5 is concise and devoted to the subject of integration:

最终我们将去往哪里！ 我们现在虽然有一些GLP (good laboratory practice)。但是还需要更多。美国和EMA发布了他们自己的指南对生物分析方法验证 in 2011。在5.5章节涉及到了积分：

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  Chromatogram integration and reintegration should be described in a SOP.
  色谱积分和重新积分应该在SOP中进行描述
  
• 
  Any deviation from this SOP should be discussed in the analytical report.
  所有偏离SOP的偏差都应该在分析报告中讨论
  
• 
  Chromatogram integration parameters and in case of re-integration, initial and the final integration data should be documented at the laboratory and should be available upon request.
  色谱积分参数和重新积分的情况，初始和最终积分数据都应该在实验室进行记录并且经过申请。
  

Although both the FDA and EMA allow reintegration of chromatograms, it must be under controlled conditions and must be reported in the final report along with who authorized the reintegration and what the original results were together with the need for audit trail entries and justification. Personally, I prefer the European approach because the FDA guidance is over-bureaucratic. This is particularly the case as the FDA requirement for documentation of the requestor and authorization by a manager will have to be outside of a CDS because there are currently no functions available to perform this. If required by regulators this needs to be included as a function in future versions of chromatography data systems.

虽然FDA和EMA允许进行色谱的重新积分，他必须在可控的条件下进行并且必须在最终报告中体现，并且在授权的情况下进行，原始数据和重新积分的数据在审计追踪中可以查到，并且进行了说明。就个人而言，我更喜欢欧洲的指南，因为FDA太过官僚。在这件事情上是个例外，FDA要求申请和管理者授权的文件可以在CDS之外，因为目前CDS还没有这个功能可以使用。如果监管者要求，将来的色谱数据系统可能会包括这个功能。

GMP办公室翻译组
译者：好累
校对：Owen

手动积分规则——手动积分还是手动干预？

GMP办公室翻译组
作者：Bob McDowall, "Questions of Quality" column editor for LCgc Europe
翻译：Lily
校对：Owen

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。

Figure 3: A suggested flow chart for manual intervention and manual integration.

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。

高效液相色谱图积分问题探讨（转载）
药群论坛 2017-09-27

高效液相色谱图怎样积分？积分规则是什么？
高效液相色谱图都是用图像处理软件来实现积分。
积分规则为：同一次测定的对照品和样品的色谱图最好是在设定一个固定的积分条件进行积分计算，积分参数一致的情况下进行可减少人为主观意识上的误差。
效液相色谱图又称“高压液相色谱”、“高速液相色谱”、“高分离度液相色谱”、“近代柱色谱”等。高效液相色谱是色谱法的一个重要分支，以液体为流动相，采用高压输液系统，将具有不同极性的单一溶剂或不同比例的混合溶剂、缓冲液等流动相泵入装有固定相的色谱柱，在柱内各成分被分离后，进入检测器进行检测，从而实现对试样的分析。
FDA对HPLC手动积分怎么看？
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）。

还有一篇无法贴上来的：
色谱积分原理和手动积分的规则WHO
http://www.docin.com/p-2023947100.html

手动积分都没有一个准确的定义，所以日常中是不是做了手动积分都不清楚，是不是工作站中归入手动积分功能的操作才叫手动积分，其它常规积分参数的调整的不算呢，还是只要导致峰基线和切割线调整就算执行了手动积分？欢迎大家各抒己见！

好帖子，先收藏，然后慢慢看

自动积分也有手动的过程，例如区分区间，最小峰面积等也是人为设定的

这就是上面提到的，是不是要设立手动积分与手动干预的区别，区间积分对于背景复杂但在计算过程中不参与结果判定时去除还是很有用的，至少可以大幅减少色谱报告的篇幅，看起来也简洁。