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1、 OMCL Network of the Council of Europe QUALITY MANAGEMENT DOCUMENT 欧洲委员会OMCL网络质量管理文件PA/PH/OMCL (11) 04QUALIFICATION OF EQUIPMENT设备的确认ANNEX 1: QUALIFICATION OF HPLC EQUIPMENT附件1：HPLC (高效液相色谱仪) 设备的确认Full document title and reference 文件全名和索引号Qualification of Equipment 设备的确认Annex 1: Qualification of HPL
2、C equipment PA/PH/OMCL (11) 04 附件1：HPLC设备的确认 PA/PH/OMCL (11) 04Document type 文件类型Guideline 指南Legislative basis 立法基础The present document was also accepted by EA as recommendation document to be used in the context of Quality Management System audits of OMCLs 本文件也被EA认可并推荐在OMCL的质量管理体系审计中使用。Date of firs
3、t adoption 首次采用日期May 2005 2005年5月Date of original entry into force 首次生效日期June 2005 2005年6月Date of entry into force of revised document 修订版生效日期1st July 2011 2011年7月1号Previous titles/other references 原文件名/其它索引号This document replaces document PA/PH/OMCL (07) 17 DEF 本文件替代文件PA/PH/OMCL (07) 17 DEFCustodia
4、n Organisation 监管组织The present document was elaborated by the OMCL Network/ EDQM of the Council of Europe 本文件由OMCL网络/欧洲EDQM制定。Concerned Network 相关网络GEON ANNEX 1 OF THE OMCL NETWORK GUIDELINE“QUALIFICATION OF EQUIPMENT”OMCL网络指南“设备的确认”之附件1QUALIFICATION OF HPLC EQUIPMENT HPLC设备的确认Introduction 概述The pre
5、sent document is the first Annex of the core document “Qualification of Equipment”, and it should be used in combination with it when planning, performing and documenting the HPLC equipment qualification process. 本文件是核心文件“设备的确认”第1个附件，在计划、实施和记录HPLC仪器的确认过程时，应将本文件与核心文件一起使用。The core document contains th
6、e general introduction and the Level I and II of qualification, common to all type of instruments, and the present annex contains HPLC instrument-related recommendations on parameters to be checked and the corresponding typical acceptance Limits, as well as practical examples on the Methodology that
7、 can be used to carry out these checks. 核心文件包括了简介和第一阶段和第二阶段的确认，它们适用于所有类型的仪器。 本附件包括了HPLC仪器相关的需要检查的参数和相应典型的可接受标准的推荐，以及可用于进行这此检查的实用方法学举例。When qualifying HPLC equipment, it should be noted that it is acceptable to check at Level III and IV several of the mentioned parameters at the same time in a combin
8、ed test procedure (e.g. “overall” system performance test giving information on peak area precision, retention time precision, gradient reproducibility, etc).在进行HPLC设备确认时，应注意也可以对第三阶段和第四阶段几个提到的参数采用联合检测程序同时进行检查（例如“全面”系统性能检查，同时给出峰面积精密度、保留时间精密度、梯度重复性等的信息）TABLE III 表三Level III. Periodic and motivated ins
9、trument checks第三阶段 定期和专门的仪器检查Examples of requirements for HPLC instruments and detectorsHPLC仪器和检测器要求举例Instrument module 仪器模块Parameter to be checked 需检查的参数Typical tolerance Limits典型误差限度 Solvent delivery system溶剂输送系统 Flow rate 流速 Proportioning accuracy and precision (gradient test) 配比准确度和精密度（梯度测试） Pro
10、portioning ripple 配比波纹5 % 2 0.2 % Injector 进样器l Volume precision 进样量精密度l Carry-over 残留l RSD1.0 % l see Annex I 见附件1Autosampler 自动进样器l Thermostatting accuracy and precision 恒温准确度和精密度l 3 C Oven or cooling device柱温箱或冷却装置 l Thermostatting accuracy 恒温准确度l 2 C UV/DAD detector UV/DAD 检测器l Linearity 线性l Wav
11、elength accuracy 波长准确度l r2 0.999 l 2 nm Fluorescence detector 荧光检测器l Wavelength accuracy excitation波长准确度激发 l Wavelength accuracy emission 波长精密度发射l Sensitivity 灵敏度l 3 nm l 3 nm l see Annex I Electrochemical detector 电化学检测器l Accuracy of the signal 信号精密度l Stability of the signal 信号稳定性l see Annex I 见附件1
12、l see Annex I 见附件1RID detector RID 检测器l Signal/Noise ratio 信噪比l Drift over time 飘移l see Annex I见附件1l 0.1 mV/min CD detector CD 检测器l Signal/Noise ratio 信噪比l Sensitivity 灵敏度l Drift over time 飘移l Linearity 线性l see Annex I见附件1l see Annex I见附件1l see Annex I见附件1l r 0.999 TABLE IV 表四Level IV. In-use instru
13、ment checks第四阶段 在用仪器的检查Examples of requirements for HPLC instruments with UV or DAD detectors配备UV或DAD检测器的HPLC仪器的要求举例Parameter to be checked 需检查参数Typical tolerance Limit 典型误差限度l System suitability check for the Method 方法系统适用性检查 According to Ph. Eur. or MAH dossier or validated in-house Method根据欧洲药典或M
14、AH文件或公司内部经过验证的方法l Peak area precision (applicable to the main peaks in the test solution) 峰面积准确度（适用于供试液中主峰面积）RSD 1.5 %(unless otherwise prescribed in the system suitability of the Method, e.g. specific requirements from Ph. Eur. 2.2.46, API monographs or MA dossiers) 除非在方法的系统适用性里有描述，例如EP2.2.46特定要求，原
15、料药各论或MA文件l Retention time precision 保留时间精密度RSD 5 %l Carry-over (by comparing consecutive standard (of the substance being quantified) and blank injections) 残留（通过比较连续（进行定量检测的物质）标准和空白进样） 0.2 % l Signal/Noise ratio (to be applied for related substances test) 信噪比（用于有关物质检测）According to Ph. Eur. 根据欧洲药典ANN
16、EX I 附件1Level III. Periodic and motivated instrument checks第三阶段 定期和专门的仪器检查This Annex contains practical examples of tests and their associated tolerance Limits for several parameters related to the performance of the different modules of a HPLC. 本附件包含的是检测HPLC仪器的不同模块性能的一些参数的实际例子及其参数的误差限度。These exampl
17、es can be considered by the OMCLs as possible approaches to perform the Level III of the equipment qualification process: “Periodic and motivated instrument checks”. OMCLs 认为这些例子可以用来执行设备确认过程的阶段III“定期和专门的仪器检测”。HPLC SOLVENT DELIVERY SYSTEM HPLC 溶剂传输系统The following tests are proposed for the periodic a
18、nd motivated check of the HPLC solvent delivery system: flow rate and gradient test. 以下测试建议用于对HPLC溶剂传输系统的周期性主动检查：流速和梯度检测FLOW RATE 流速Materials: 材料Volumetric flask of 5 or 10 ml 5ml或10ml的容量瓶Calibrated chronometer 校正过的气压计Settings: 设置Mobile phase: degassed water 流动相：脱过气的水No column (open end)* 无柱（开放式）*Fl
19、ow rate: adjusted between 0.5 and 3.0 ml/min 流速：调整到0.5至3.0ml/min之间If high-pressure mixing systems are installed, this test has to be done on each solvent channel. 如果有安装高压混合系统，本测试应在每一溶剂通道进行。* For certain equipment, e.g. in the case of low flow rates, the check would be performed by using a column or
20、a backpressure regulator. * 对于某些设备，例如，流速较低时，检测时需要用到一根色谱柱或背压调节器进行检查。Method方法: Set the flow rate at an appropriate level and measure the time needed to fill the volumetric flask up to the mark. Record the time needed. 将流速设定在一个合适的水平，测量将容量瓶装满至刻度的时间。记录所用的时间。 f V /t f (V60)/tf . measured flow rate ml/min
21、测得的流速 t . elapsed time to fill up to mark s 充满至刻度所需的时间V . volume of the volumetric flask ml 容量瓶的体积 D100(fF)/FD . deviation % 偏差F . adjusted flow rate ml/min 调节后的流速f . measured flow rate ml/min 测量的流速Limits限度: 5%GRADIENT COMPOSITION AND RIPPLE 梯度组成和波动Settings: 设置Stainless steel capillary e.g. 2000 x 0
22、.12 mm installed instead of a column 不锈钢毛细管，例如2000X0.12mm，代替色谱柱安装于仪器Detection: UV-Detector adjusted to 265 nm 检测器：紫外检测器调节至265nmMobile phase A: degassed water 流动相A：脱气水Mobile phase B: degassed water containing 0.5% acetone 流动相B：含0.5%的脱气水Flow rate: 1.0 ml/min 流速：1.0ml/分钟Method方法: The test is carried ou
23、t in the following way by using a gradient program depending on the number of solvent channels and the configuration of the system: 根据溶剂通道的数量和系统的配置，使用下列不同系统参数和梯度进行测试：A-B A-B and A-C A-C, A-B and B-Dtime min时间【分钟】% mobile phase A (water)%流动相A（水）% mobile phase B% 流动相B(water-acetone mixture)（水-丙酮混合物）0.
24、010000.1901010901010.1505020505020.1109030109030.1010040010040.11000Start the test by pumping water for at least 10 min to equilibrate the system. 开始测试前，先用水平衡系统至少10分钟。The zero % value at the start of the test is the baseline. All steps are measured at the beginning of the horizontal part of the line
25、 either by software or manually on the paper print using a liner. The height of the 100% water/acetone mixture is used as the 100% value in the following calculation. 测试开始时的0%值是基线值。所有步骤均在基线的水平部分开始时由软件或人工测量，并打印在纸上。100%水/丙酮混合物的高度在以下计算中作为100%值。 %H100* h/H %H . calculated composition 计算组成h . height of t
26、he measured line 测量线的高度H . height of the 100% water/acetone mixture line (mobile phase B) 100%水/丙酮混合物线的高度（流动相B） d %HG d . deviation 偏差G . gradient composition adjusted % acetone/water solution mixture = mobile phase B 梯度组成调节【%丙酮/水溶液混合物 = 流动相B】Limits限度: Absolute deviation: 2 of the adjusted value 绝对偏
27、差： 调整值的2The ripple of the gradient composition is the percentage of noise of the 50% line from the gradient program. 梯度组分的波动为梯度程序噪声基线的50% %R 100* N /h50 %R . ripple 波纹h50 . height of the 50% line 50%线的高度N . height of the noise line, measured during 1 minute in the linear region 噪声线高度，在线性区域1分钟期间测量的结果
28、Limits 限度: 0.2 % HPLC INJECTOR HPLC进样器Volume precision and carry-over are the tests proposed for the periodic and motivated check of the HPLC injector. 建议在HPLC进样器的定期和专门检查中进行容量(体积)精密度和残留检查。VOLUME PRECISION AND CARRY-OVER 体积精密度和残留Solutions: 溶液Solvent A: methanol : water R, 60 : 40. 溶液 A：乙醇：水（试剂） = 60：
29、40Reference solution (a): dissolve 15.0 mg methyl-, ethyl-, and propylparabene in 100.0 ml of solvent A. 对照液（a）：取15.0mg尼泊金M、尼泊金乙酯和尼泊金丙酯溶于100.0ml的溶剂A中Reference solution (b): Dilute 1.0 ml of reference solution (a) to 10.0 ml of solvent A. 对照液（b）：取1.0ml对照液（a）用溶于10.0ml溶剂A中Reference solution (c): Dilute
30、 1.0 ml of reference solution (b) to 100.0 ml of solvent A. 对照液（c）：取1.0ml对照液（b）用溶于100.0ml溶剂A中Settings: 设置Column: Lichrospher 100 RP8, 5um, 125 x 4 mm, without precolumn 色谱柱：Lichrospher 100 RP8, 5 um, 125 x 4 mm, 无衬管Mobile phase: methanol : water = 60 : 40 流动相：乙醇：水 = 60：40Flow rate: 1.0 ml/min 流速：1.0
31、ml/分钟Detection: 254 nm 检测器：254nmInjection volume: 20 l 进样体积：20ulMethod方法: Injection scheme: 进样序列 6x reference solution (b) 对照液（b）6针 1x reference solution (a) 对照液（a）1针 1x solvent A (blank injection 1) 溶剂A（空白1）1针 1x reference solution (b) 对照液（b）1针 1x solvent A (blank injection 2) 溶剂A（空白2）1针 1x referen
32、ce solution (c) 对照液（c）1针Limit限度s: Repeatability of peak areas: The relative standard deviation of the peak areas of all peaks in the chromatogram obtained with the reference solution (b) should be 1.0 %.峰面积重复性：对照液（b）色谱图中所有峰的峰面积相对标准偏差应1.0 %Carry-over: The percentage of the peak area corresponding to
33、propylparabene in the blank injection 1 does not exceed 0.5% of 10 times the peak area of the propylparabene peak in the chromatogram obtained with the reference solution (b) injected after the blank injection. 残留：尼泊金丙酯在空白图谱中的峰面积不超过在空白后进样的对照液（b）中尼泊金丙酯的对应峰面积的10倍的0.5%The percentage of the peak area co
34、rresponding to propylparabene in the reference solution (c) is 0.9 1.1% of the peak area of the propylparabene peak in the chromatogram obtained with the reference solution (b) injected after the blank injection. 在对照液（c）中尼泊金丙酯对应的峰面积为空白后进样的对照液（b）中尼泊金丙酯峰面积的0.9-1.1%。HPLC AUTOSAMPLER HPLC 自动进样器Thermosta
35、tting accuracy and precision can be tested in the frame of the periodic and motivated check of the HPLC Autosampler. 对HPLC自动进样器的定期和专门检查可以检测温度的准确度和精确度。THERMOSTATTING ACCURACY 温度准确度Materials: 材料Calibrated temperature probe. 校正过的温度探头Method方法: Select a temperature along the operational or required tempe
36、rature range of the equipment. Wait until the system is equilibrated. 在可操作范围或要求的温度范围内选择并设定一个温度，等待系统平衡By means of the calibrated probe, measure the actual temperature in the autosampler and compare it to the selected temperature. 采用校正过的温度探头，测量自动进样器的实际温度，与设定的温度进行比较Repeat the same procedure at differen
37、t pre-selected points covering the temperature range. 在选定的温度范围内的不同温度点重复相同的步骤Limit限度s: The actual temperature may not differ more than 3C with respect to the selected temperature. 实际温度与设定的温度相比差值不超过3CTHERMOSTATTING PRECISION 温度精确度Materials: 材料Calibrated temperature probe. 校正过的温度探头。Method方法: Select a t
38、emperature along the operational or required temperature range of the equipment. Wait until the system is equilibrated. 选择一个操作温度或要求的仪器温度范围，等待系统平衡。By means of the calibrated probe, make “n” measurements over a pre-established period of time. Compare the mean of the “n” measurements to the selected te
39、mperature. 采用校正过的温度探头，在预设的时间内进行n次测量，比较n次测量值的平均值与设定的温度。Limits限度: The actual temperature may not differ more than 3C with respect to the selected temperature.实际温度与设定的温度之间差值不超过3CHPLC OVEN/COOLING DEVICE 色谱仪柱温箱/降温设施Thermostatting accuracy is the parameter tested in this example of periodic and motivated
40、 check of the HPLC oven/cooling device. 温度的准确度是对HPLC柱温箱/降温设施进行定期和专门检查时检测的参数。THERMOSTATTING ACCURACY 温度准确性Materials: 材料Calibrated thermometer. 校正过的温度计Method方法: Set the column oven temperature to 40 C, wait about 30 minutes to equilibrate the system, put a calibrated thermometer into the oven and read
41、 the temperature after 10 minutes. 设定柱温箱温度为40 C，等待约30分钟平衡系统，将校正过的温度计放入柱温箱中，在10分钟后读取温度。Limits限度: 38 - 42C. HPLC UV/DAD DETECTOR HPLC 紫外/DAD检测器The periodic and motivated check of the HPLC UV/DAD detector can be performed by testing the linearity and the wavelength accuracy. 对HPLC紫外/DAD检测器的定期和专门的检查，可以对
42、线性和波长准确性进行检测。LINEARITY 线性Solutions: 溶剂配制Std. 1: 0.5 g caffeine/1 ml methanol HPLC Grade 标准1： 0.5 g咖啡因/1ml色谱级甲醇Std. 2: 1.0 g caffeine/1 ml methanol HPLC Grade 标准2： 1.0 g咖啡因/1ml色谱级甲醇Std. 3: 5.0 g caffeine/1 ml methanol HPLC Grade 标准3： 5.0 g咖啡因/1ml色谱级甲醇Std. 4: 25.0 g caffeine/1 ml methanol HPLC Grade 标
43、准4：25.0 g咖啡因/1ml色谱级甲醇Std. 5: 50.0 g caffeine/1 ml methanol HPLC Grade 标准5：50.0 g咖啡因/1ml色谱级甲醇Std. 6: methanol HPLC Grade (blank) 标准6：色谱级甲醇（空白）Std. 5: weigh 9.0 to 11.0 mg caffeine and fill up to 200.0 ml with methanol HPLC 标准5：称取9.0至11.0mg咖啡因，用HPLC级别甲醇稀释至200.0mlStd. 4: dilute 50.0 ml of Std. 5 to 100
44、.0 ml with methanol 标准4：取50.0ml标准5溶液用甲醇稀释至100.0mlStd. 3: dilute 10.0 ml of Std. 5 to 100.0 ml with methanol 标准3：取10.0ml标准溶液5用甲醇稀释至100.0mlStd. 2: dilute 20.0 ml of Std. 3 to 100.0 ml with methanol 标准2：取20.0ml标准3溶液用甲醇稀释至100.0mlStd. 1: dilute 10.0 ml of Std. 3 to 100.0 ml with methanol 标准1：取10.0ml标准3用甲
45、醇稀释至100.0mlSettings: 设置Column: RP-18 5 um 30-50 x 2,1-4.6 mm or capillary 2000 mm x 0.12 mm ID 色谱柱：RP-185 um 30-50 x 2,1-4.6 mm or 毛细管柱 2000 mm x 0.12 mm IDMobile phase: methanol HPLC Grade 流动相：色谱级甲醇Oven temperature: 40 C 柱温箱温度：40 CFlow rate: 1.0 ml/min (adjusted by using 100% methanol) 流速：1.0ml/min（用100%甲醇调整）Detection: 273 nm 检测波长：273nmInjection volume: 20l进样量：20ulMethod方法: Injection scheme: 进样序列2 x blank 2针空白1 x Std. 1 1 x Std. 2 1 x Std. 31 x Std. 41 x Std. 5 标
