色谱积分原理和手动积分的规则
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 en during an inspection, you can almost e 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 obrvation 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 rious 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.
50字小故事此外，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。人穷怪屋基
There have been many other cas 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 t correctly and the chromatography is acceptable then there should be no need to reintegrate manually in many cas. However, we also need to acknowledge that chromatographic systems are dynamic by their nature and parations 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-t. Although the focus of this column is manual integration plea do not lo sight of the bigger picture. As such, we w
油鸱ill 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 becau the latter has been covered in an earlier Questions of Quality column . Furthermore we will not be considering the u of system suitability tests (SSTs), again becau this column has already discusd 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
隋唐演义读后感色谱积分基础
吉他琴谱
Before we can discuss how to control and manage manual integration it is important to understand the basics of chromatographic integration itlf. The best book on the subject of integration is by Norman Dyson and, although the cond 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 discusd below. Note that some CDS suppliers may call the parameters a different name but the functionality is esntially 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
汉堡英文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 fi
le 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. The 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 noi to allow better peak measurement .
一旋一柱思华年
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。
好心情Peak width or sampling rate tting 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. Converly, 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.
