欧盟OMCL仪器确认附件1-HPLC确认

OMCL Network of the Council of EuropeQUALITY MANAGEMENT DOCUMENTPA/PH/OMCL (11) 04QUALIFICATION OF EQUIPMENT仪器确认ANNEX 1: QUALIFICATION OF HPLC EQUIPMENT附件1：HPLC仪器的确认Full document title and reference文件全名和索引号Qualification of EquipmentAnnex 1: Qualification of HPLC equipmentPA/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 OMCLsDate of first adoption首次发行日期May 2005Date of original entry into force首次执行日期June 2005Date of entry into force of revised document 修订后执行日期1st July 2011Previous titles/other references原文件名/其它索引号This document replaces document PA/PH/OMCL (07) 17 DEFCustodian Organisation保管机构The present document was elaborated by the OMCL Network/ EDQM of the Council of EuropeConcerned Network相关网络GEONANNEX 1 OF THE OMCL NETWORK GUIDELINE“QUALIFICATION OF EQUIPMENT”OMCL网络指南“仪器的确认”之附件1QUALIFICATION OF HPLC EQUIPMENTHPLC仪器的确认Introduction 概述The present 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“仪器的确认”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 the 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 can be used to carry out these checks.核心文件包括了第一级和第二级确认的通用介绍，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 combined test procedure (e.g. “overall” system performance test giving information on peak area precision, retention time precision, gradient reproducibility, etc).在进行HPLC仪器确认时，应注意也可以对第三级和第四级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 instrument checks第三级 定期主动仪器检查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典型允差限度Solvent delivery system溶剂传送系统l Flow rate 流速l Proportioning accuracy and precision (gradient test) 配比准确度和精密度（梯度测试）l Proportioning ripple 配比波动l 5 %l 2l 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Thermostatting accuracy and precision 恒温准确度和精密度l 3 COven or cooling device柱温箱或冷却装置Thermostatting accuracy恒温准确度l 2 CUV/DAD detectorUV/DAD检测器l Linearity 线性l Wavelength accuracy波长准确度l r2 0.999l 2 nmFluorescence detector荧光检测器l Wavelength accuracy excitation波长激发准确度l Wavelength accuracy emission 波长发射准确度l Sensitivity 灵敏度l 3 nml 3 nml see Annex IElectrochemical detector 电化学检测器l Accuracy of the signal 信号准确度l Stability of the signal 信号稳定性l see Annex I 见附件1l see Annex I 见附件1RID detectorRID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/minCD detectorCD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 0.999TABLE IV 表四Level IV. In-use instrument checks第四级 在用仪器检查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 Limits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MAH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ANNEX I 附件1Level III. Periodic and motivated instrument checks第三级 定期性的主动仪器检查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.本附件包括一些实用几个与HPLC不同模块相关的参数检测举例及其相应的可接受限度。These examples can be considered by the OMCLs as possible approaches to perform the Level III of the equipment qualification process: “Periodic and motivated instrument checks”.这些例子可以认为是OMCL实验室可能的进行第三级仪器确认的方法“周期主动仪器检查”HPLC SOLVENT DELIVERY SYSTEM 高压液相溶剂传输系统The following tests are proposed for the periodic and motivated check of the HPLC solvent delivery system: flow rate and gradient test.以下测试作为HPLC溶剂传输系统周期性主动检查：流速和梯度检查FLOW RATE 流速Materials: 材料Volumetric flask of 5 or 10 ml5ml或10ml的容量瓶Calibrated chronometer校正过的计时计Settings: 设置Mobile phase: degassed water流动相：脱过气的水No column (open end)*无柱（开放式）*Flow rate: adjusted between 0.5 and 3.0 ml/min流速：调节在0.5到3.0ml/minIf 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。T For certain equipment, e.g. in the case of low flow rates, the check would be performed by using a column or a backpressure regulator.对于一定的仪器，例如，流速较低时，采用一根柱子或背压调整器进行检查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 测量的流速t . elapsed time to fill up to mark s 充满至刻度所花的时间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 0.12 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%丙酮的脱气水Flow rate: 1.0 ml/min流速：1.0ml/分钟Method方法:The test is carried out in the following way by using a gradient program depending on the number of solvent channels and the configuration of the system:按下列方法进行测试，根据通道数量不同使用下列不同系统参数和梯度进行洗脱A-BA-B and A-CA-C, A-B and B-Dtime min时间分钟% mobile phase A (water)%流动相A（水相）% mobile phase B %流动相B(water-acetone mixture)（水-丙酮混合物）0.010000.1901010901010.1505020505020.1109030109030.1010040010040.11000Start 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 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.开始时的0%值是基线值。所有步骤均在开始时测量基线的水平部分，由软件或手动在纸上用衬垫打印。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 the 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 %HGd . 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%的线高度N . height of the noise line, measured during 1 minute in the linear reg ion 噪声线高度，在线性区域1分钟测量的结果 Limit限度s: 0.2 %HPLC INJECTOR 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进样器周期性主动检查中进行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） = 60：40Reference solution (a): dissolve 15.0 mg methyl-, ethyl-, and propylparabene in 100.0 ml of solvent A.对照液（a）：取15.0mg乙基、甲基和尼泊金丙酯溶于100.0ml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 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流动速度：1.0ml/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 reference solution (c) 对照液（c）1针Limits限度: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 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 corresponding 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 液相色谱仪自动进样器Thermostatting accuracy and precision can be tested in the frame of the periodic and motivated check of the HPLC Autosampler.HPLC自动进样器的周期主动检查可以进行温度准确度和精密度检查THERMOSTATTING ACCURACY 温度准确性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 temperature 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 different pre-selected points covering the temperature range.在选定的温度范围内不同温度点重复相同的步骤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相比差值不超过3CTHERMOSTATTING PRECISION 温度准确性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 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 temperature.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 check of the HPLC oven/cooling device.热力学准确性是在本例对HPLC柱温箱/降温设施周期主动检查的参数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 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 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.对于HPLC紫外/DAD检测器的周期主动检查，可以对线性和波长准确性进行检查LINEARITY 线性Solutions: 溶剂配制Std. 1: 0.5 g caffeine/1 ml methanol HPLC Grade标准2： 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 100.0 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-18 5 um 30-50 x 2,1-4.6 mm or capillary 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 blank1 x Std. 11 x Std. 21 x Std. 31 x Std. 41 x Std. 52针空白，标准1-5依序各一针Limits限度: r2 0.999Remark: As this test employs different test solutions to be injected, it covers also the check of correct positioning vials in the autosampler.注：由于此检测中要注入不同溶液，因此也包括了自动进样器样品瓶位置正确性的确认WAVELENGTH ACCURACY 波长准确度If there are built-in test procedures for the determination and adjustment of wavelength accuracy, follow the instructions of the instrument manual.如果内置检测程序可以检测和调节波长准确度，则根据操作手册进行操作In all other cases use the procedure described below.在所有其它情况下采用以下程序Solutions: 溶液DAD: caffeine Std. 5 from the linearity testing 线性测试中咖啡因标准溶液5号UV/VIS: 1.0 g/ml anthracene solution in water 蒽水溶液Settings: 设置Mobile phase: 15% acetonitrile in water流动相：15%乙腈水溶液Column: RP18, 5 ?m 30-50 x 2,1-4.6 mm or capillary 2.0 m x 0.12 mm ID色谱柱：RP18，5m, 30-502.1-4.6mm或毛细管柱20m0.12mm内径Oven temperature: 40 C柱箱温度：40Flow rate: 1.0 ml/min (adjusted by using 15% acetonitrile in water)流速：1.0ml/min（用15%的乙腈水溶液调节）Detection: scan from 230 nm to 290 nm (DAD)检测：自230至290nm扫描（DAD）Injection volume: 20 l进样体积：20lMethod方法:DAD: Inject 20l of the caffeine solution and record the spectrum. The maximum is at 272 nm and the minimum at 244 nm.DAD: 咖啡因溶液进样20l，记录色谱图，最大吸收波长为272nm，最小为244nm.UV/VIS: fill the cell with the anthracene solution and change the wavelength from 248 to 254 nm in 1 nm steps; record the maximum of absorption. The theoretical value is 251 nm.UV/可见光：向池中注入蒽溶液，将波长以1nm的步距从248变化至254nm，记录最大吸收值。理论值为251nm。Limit限度s: 2 nmHPLC FLUORESCENCE DETECTOR 荧光检测器The following three parameters are proposed for the performance of the periodic and motivated check of the HPLC fluorescent detector:下面三个参数建议作为荧光检测器性能周期自主检查WAVELENGTH ACCURACY EXCITATION 波长激发准确度Method: 方法Rinse and fill the measuring cell with de-ionized water用去离子水淋洗并充满检测池Adjust the excitation wavelength to 350 nm.调节激发波长为350nmMeasure the emission and subtract 397 nm (theoretical value).测量发射波长减去397nm（理论值）Limits限度: 3 nmWAVELENGTH ACCURACY EMISSION 波长发射准确度Method: 方法Rinse and fill the measuring cell with de-ionized water用去离子水淋洗并充满检测池Adjust the emission wavelength to 397 nm.调节发射波长为397nmMeasure the excitation and subtract 350 nm (theoretical value).测量激发波长减去350nm（理论值）Limits限度:3nmSENSITIVITY 灵敏度Solutions: 溶液Quinine HCl.2H2O solution conc. 0.015g/ml (=15 ppb)二水盐酸喹啉溶液，浓度0.015g/ml (=15 ppb)The quinine solution is prepared with the following mobile phase: dissolve 6.8 g of potassium dihydrogen phosphate R and 3.0 g of hexylamine R in 700 ml of water R, adjust to pH 2.8 with dilute phosphoric acid R, add 90 ml of acetonitrile R and dilute to 1000.0 ml with water R.喹啉溶液用以下流动相制备：溶解6.8g磷酸二氢钾和3.0g己胺于700ml试剂级水中，用稀磷酸调pH至2.8，加90ml乙腈并用试剂级水稀释至1000.0ml.Settings: 设置The chromatographic conditions are set according to Ph. Eur. “Quinine HCl” (01/2005:0018), test “Other cinchona alkaloids”, with modified flow rate and acetonitrile concentration. 色谱条件设定依据欧洲药典“盐酸喹啉”（01/2005:0018），检测“金鸡纳生物碱”，改变流速和乙腈浓度。Mobile phase: as above流动相：同上Column: RP18, 5 m, 250 4.6 mm色谱柱: RP18, 5 m, 250 4.6 mmFlow rate: 1.2 ml/min流速：1.2 ml/minExcitation wavelength: 350 nm激发波长：350nmEmission wavelength: 397 nm发射波长：397nmFlow-cell volume: 8 l (for this example, a Waters 2475 Multi Fluorescentie detector was used. Flow-cell volume may vary depending on the instrument manufacturer)流动池体积：8l（此例中，使用的是Waters2475多荧光检测器，流动池体积可能根据仪器厂商不同而不同）Method方法:Inject 10l of the quinine solution and measure the peak