手动积分原则及SOP！

GMP办公室翻译组

翻译：科学的mushroom

校对：Owen

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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。

手动积分申请及处理审批表示例如下：