欧盟OMCL仪器确认附件1-HPLC确认

OMCLNetworkoftheCouncilofEuropeQUALITYMANAGEMENTDOCUMENTPA/PH/OMCL(11)04QUALIFICATIONOFEQUIPMENT仪器确认ANNEX1:QUALIFICATIONOFHPLCEQUIPMENT附件1：HPLC仪器的确认Fulldocumenttitleandreference文件全名和索引号QualificationofEquipmentAnnex1:QualificationofHPLCequipmentPA/PH/OMCL(11)04Documenttype文件类型Guideline指南Legislativebasis立法基础ThepresentdocumentwasalsoacceptedbyEAasrecommendationdocumenttobeusedinthecontextofQualityManagementSystemauditsofOMCLsDateoffirstadoption首次发行日期May2005Dateoforiginalentryintoforce首次执行日期June2005Dateofentryintoforceofreviseddocument修订后执行日期1stJuly2011Previoustitles/otherreferences原文件名/其它索引号ThisdocumentreplacesdocumentPA/PH/OMCL(07)17DEFCustodianOrganisation保管机构ThepresentdocumentwaselaboratedbytheOMCLNetwork/EDQMoftheCouncilofEuropeConcernedNetwork相关网络GEON  ANNEX1OFTHEOMCLNETWORKGUIDELINE“QUALIFICATIONOFEQUIPMENT”OMCL网络指南“仪器的确认”之附件1QUALIFICATIONOFHPLCEQUIPMENTHPLC仪器的确认Introduction概述ThepresentdocumentisthefirstAnnexofthecoredocument“QualificationofEquipment”,anditshouldbeusedincombinationwithitwhenplanning,performinganddocumentingtheHPLCequipmentqualificationprocess.本文件是核心文件“仪器的确认”第1个附件，在 计划 项目进度计划表范例计划下载计划下载计划下载课程教学计划下载 、实施和 记录 混凝土 养护记录下载土方回填监理旁站记录免费下载集备记录下载集备记录下载集备记录下载 HPLC仪器的确认过程时，应将本文件与核心文件一起使用。 ThecoredocumentcontainsthegeneralintroductionandtheLevelIandIIofqualification,commontoalltypeofinstruments,andthepresentannexcontainsHPLCinstrument-relatedrecommendationsonparameterstobecheckedandthecorrespondingtypicalacceptanceLimits,aswellaspracticalexamplesontheMethodologythatcanbeusedtocarryoutthesechecks.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 excel标准偏差excel标准偏差函数exl标准差函数国标检验抽样标准表免费下载红头文件格式标准下载 的推荐，以及可用于进行这此检查的实用方法学举例。 WhenqualifyingHPLCequipment,itshouldbenotedthatitisacceptabletocheckatLevelIIIandIVseveralofthementionedparametersatthesametimeinacombinedtestprocedure(e.g.“overall”systemperformancetestgivinginformationonpeakareaprecision,retentiontimeprecision,gradientreproducibility,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）TABLEIII表三LevelIII.Periodicandmotivatedinstrumentchecks第三级定期主动仪器检查ExamplesofrequirementsforHPLCinstrumentsanddetectorsHPLC仪器和检测器要求举例Instrumentmodule仪器模块Parametertobechecked需检查的参数TypicaltoleranceLimits典型允差限度Solventdeliverysystem溶剂传送系统Flowrate流速Proportioningaccuracyandprecision(gradienttest)配比准确度和精密度（梯度测试）Proportioningripple配比波动±5%±2≤0.2%Injector进样器Volumeprecision进样量精密度Carry-over残留RSD≤1.0%seeAnnexI见附件1Autosampler自动进样器Thermostattingaccuracyandprecision恒温准确度和精密度±3°COvenorcoolingdevice柱温箱或冷却装置Thermostattingaccuracy恒温准确度±2°CUV/DADdetectorUV/DAD检测器Linearity线性Wavelengthaccuracy波长准确度r2≥0.999±2nmFluorescencedetector荧光检测器Wavelengthaccuracyexcitation波长激发准确度Wavelengthaccuracyemission波长发射准确度Sensitivity灵敏度±3nm±3nm±seeAnnexIElectrochemicaldetector电化学检测器Accuracyofthesignal信号准确度Stabilityofthesignal信号稳定性seeAnnexI见附件1seeAnnexI见附件1RIDdetectorRID检测器Signal/Noiseratio信噪比Driftovertime飘移seeAnnexI见附件10.1mV/minCDdetectorCD检测器Signal/Noiseratio信噪比Sensitivity灵敏度Driftovertime飘移Linearity线性seeAnnexI见附件1seeAnnexI见附件1seeAnnexI见附件1r>0.999 TABLEIV表四LevelIV.In-useinstrumentchecks第四级在用仪器检查ExamplesofrequirementsforHPLCinstrumentswithUVorDADdetectors配备UV或DAD检测器的HPLC仪器的要求举例Parametertobechecked需检查参数TypicaltoleranceLimits限度SystemsuitabilitycheckfortheMethod方法系统适用性检查AccordingtoPh.Eur.orMAHdossierorvalidatedin-houseMethod根据欧洲药典或MAH文件或公司内部经过验证的方法Peakareaprecision(applicabletothemainpeaksinthetestsolution)峰面积准确度（适用于供试液中主峰面积）RSD≤1.5%(unlessotherwiseprescribedinthesystemsuitabilityoftheMethod,e.g.specificrequirementsfromPh.Eur.2.2.46,APImonographsorMAdossiers)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Retentiontimeprecision保留时间精密度RSD±5%Carry-over(bycomparingconsecutivestandard(ofthesubstancebeingquantified)andblankinjections)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%Signal/Noiseratio(tobeappliedforrelatedsubstancestest)信噪比（用于有关物质检测）AccordingtoPh.Eur.根据欧洲药典ANNEXI附件1LevelIII.Periodicandmotivatedinstrumentchecks第三级定期性的主动仪器检查ThisAnnexcontainspracticalexamplesoftestsandtheirassociatedtoleranceLimitsforseveralparametersrelatedtotheperformanceofthedifferentmodulesofaHPLC.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。TheseexamplescanbeconsideredbytheOMCLsaspossibleapproachestoperformtheLevelIIIoftheequipmentqualificationprocess:“Periodicandmotivatedinstrumentchecks”.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” HPLCSOLVENTDELIVERYSYSTEM高压液相溶剂传输系统ThefollowingtestsareproposedfortheperiodicandmotivatedcheckoftheHPLCsolventdeliverysystem:flowrateandgradienttest.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FLOWRATE流速Materials:材料Volumetricflaskof5or10ml5ml或10ml的容量瓶Calibratedchronometer校正过的计时计 Settings:设置Mobilephase:degassedwater流动相：脱过气的水Nocolumn(openend)*无柱（开放式）*Flowrate:adjustedbetween0.5and3.0ml/min流速：调节在0.5到3.0ml/minIfhigh-pressuremixingsystemsareinstalled,thistesthastobedoneoneachsolventchannel.如果有安装高压混合系统，本测试应在每一溶剂通道进行。Forcertainequipment,e.g.inthecaseoflowflowrates,thecheckwouldbeperformedbyusingacolumnorabackpressureregulator.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:Settheflowrateatanappropriatelevelandmeasurethetimeneededtofillthevolumetricflaskuptothemark.Recordthetimeneeded.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/tf＝(V×60)/tf.................measuredflowrate[ml/min]测量的流速t.................elapsedtimetofilluptomark[s]充满至刻度所花的时间V...............volumeofthevolumetricflask[ml]容量瓶的体积D＝100×(f－F)/FD...............deviation[%]偏差F...............adjustedflowrate[ml/min]调节后的流速f.................measuredflowrate[ml/min]测量的流速Limits限度:±5% GRADIENTCOMPOSITIONANDRIPPLE梯度组成和波动 Settings:设置Stainlesssteelcapillarye.g.2000x0.12mminstalledinsteadofacolumn不锈钢毛细管，例如2000X0.12mm，代替色谱柱安装于仪器Detection:UV-Detectoradjustedto265nm检测器：紫外检测器调节至265nmMobilephaseA:degassedwater流动相A：脱气水MobilephaseB:degassedwatercontaining0.5%acetone流动相B：含0.5%丙酮的脱气水Flowrate:1.0ml/min流速：1.0ml/分钟 Method方法:Thetestiscarriedoutinthefollowingwaybyusingagradientprogramdependingonthenumberofsolventchannelsandtheconfigurationofthesystem: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-BandA-CA-C,A-BandB-Dtime[min]时间[分钟]%mobilephaseA(water)%流动相A（水相）%mobilephaseB%流动相B(water-acetonemixture)（水-丙酮混合物）0.010000.1901010901010.1505020505020.1109030109030.1010040010040.11000Startthetestbypumpingwaterforatleast10mintoequilibratethesystem.开始测试前，先用水平衡系统至少10分钟。Thezero%valueatthestartofthetestisthebaseline.Allstepsaremeasuredatthebeginningofthehorizontalpartofthelineeitherbysoftwareormanuallyonthepaperprintusingaliner.Theheightofthe100%water/acetonemixtureisusedasthe100%valueinthefollowingcalculation.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。%H＝100*h/H%H............calculatedcomposition计算组成h................heightofthemeasuredline测量线的高度H...............heightofthe100%water/acetonemixtureline(mobilephaseB) 100%水/丙酮混合物线的高度（流动相B）d＝%H－Gd................deviation偏差G...............gradientcompositionadjusted[%acetone/watersolutionmixture=mobilephaseB] 梯度组成调节【%丙酮/水溶液混合物=流动相B】 Limits限度:Absolutedeviation:±2oftheadjustedvalue绝对偏差：调整值的±2Therippleofthegradientcompositionisthepercentageofnoiseofthe50%linefromthegradientprogram.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.............heightofthe50%line50%的线高度N...............heightofthenoiseline,measuredduring1minuteinthelinearregion噪声线高度，在线性区域1分钟测量的结果 Limit限度s:≤0.2% HPLCINJECTOR进样器Volumeprecisionandcarry-overarethetestsproposedfortheperiodicandmotivatedcheckoftheHPLCinjector.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VOLUMEPRECISIONANDCARRY-OVER体积精密度和残留Solutions:溶液SolventA:methanol:waterR,60:40.溶液A：乙醇：水（试剂）=60：40Referencesolution(a):dissolve15.0mgmethyl-,ethyl-,andpropylparabenein100.0mlofsolventA.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Referencesolution(b):Dilute1.0mlofreferencesolution(a)to10.0mlofsolventA.对照液（b）：取1.0ml对照液（a）用溶于10.0ml溶剂A中Referencesolution(c):Dilute1.0mlofreferencesolution(b)to100.0mlofsolventA.对照液（c）：取1.0ml对照液（b）用溶于100.0ml溶剂A中 Settings:设置Column:Lichrospher100RP8,5um,125x4mm,withoutprecolumn色谱柱：Lichrospher100RP8,5um,125x4mm,无衬管Mobilephase:methanol:water=60:40流动相：乙醇：水=60：40Flowrate:1.0ml/min流动速度：1.0ml/分钟Detection:254nm检测器：254nmInjectionvolume:20μl进样体积：20ul Method方法:Injectionscheme:进样序列—       6xreferencesolution(b)对照液（b）6针—      1xreferencesolution(a)对照液（a）1针—       1xsolventA(blankinjection1)溶剂A（空白）1针—       1xreferencesolution(b)对照液（b）1针—       1xsolventA(blankinjection2)溶剂A（空白2）1针—       1xreferencesolution(c)对照液（c）1针 Limits限度:Repeatabilityofpeakareas:Therelativestandarddeviationofthepeakareasofallpeaksinthechromatogramobtainedwiththereferencesolution(b)shouldbe≤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:Thepercentageofthepeakareacorrespondingtopropylparabeneintheblankinjection1doesnotexceed0.5%of10timesthepeakareaofthepropylparabenepeakinthechromatogramobtainedwiththereferencesolution(b)injectedaftertheblankinjection.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% Thepercentageofthepeakareacorrespondingtopropylparabeneinthereferencesolution(c)is0.9–1.1%ofthepeakareaofthepropylparabenepeakinthechromatogramobtainedwiththereferencesolution(b)injectedaftertheblankinjection.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%。 HPLCAUTOSAMPLER液相色谱仪自动进样器 ThermostattingaccuracyandprecisioncanbetestedintheframeoftheperiodicandmotivatedcheckoftheHPLCAutosampler.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 THERMOSTATTINGACCURACY温度准确性Materials:材料Calibratedtemperatureprobe.校正过的温度探头 Method方法:Selectatemperaturealongtheoperationalorrequiredtemperaturerangeoftheequipment.Waituntilthesystemisequilibrated.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Bymeansofthecalibratedprobe,measuretheactualtemperatureintheautosamplerandcompareittotheselectedtemperature.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Repeatthesameprocedureatdifferentpre-selectedpointscoveringthetemperaturerange.在选定的温度范围内不同温度点重复相同的步骤 Limits限度:Theactualtemperaturemaynotdiffermorethan±3°Cwithrespecttotheselectedtemperature.实际温度与设定的温度相比差值不超过±3°C THERMOSTATTINGPRECISION温度准确性 Materials:材料Calibratedtemperatureprobe.校正过的温度探头。 Method方法:Selectatemperaturealongtheoperationalorrequiredtemperaturerangeoftheequipment.Waituntilthesystemisequilibrated.选择一个操作温度或要求的仪器温度范围，等待系统平衡。Bymeansofthecalibratedprobe,make“n”measurementsoverapre-establishedperiodoftime.Comparethemeanofthe“n”measurementstotheselectedtemperature.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:Theactualtemperaturemaynotdiffermorethan±3°Cwithrespecttotheselectedtemperature.实际温度与设定的温度之间差值不超过±3°CHPLCOVEN/COOLINGDEVICE 色谱仪柱温箱/降温设施 ThermostattingaccuracyistheparametertestedinthisexampleofperiodicandmotivatedcheckoftheHPLCoven/coolingdevice.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 THERMOSTATTINGACCURACY温度准确性 Materials:材料Calibratedthermometer.校正过的温度计 Method方法:Setthecolumnoventemperatureto40°C,waitabout30minutestoequilibratethesystem,putacalibratedthermometerintotheovenandreadthetemperatureafter10minutes.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:38-42°C. HPLCUV/DADDETECTOR紫外/DAD检测器TheperiodicandmotivatedcheckoftheHPLCUV/DADdetectorcanbeperformedbytestingthelinearityandthewavelengthaccuracy.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.1:0.5μgcaffeine/1mlmethanolHPLCGrade标准2：0.5μg咖啡因/1ml色谱级甲醇Std.2:1.0μgcaffeine/1mlmethanolHPLCGrade标准2：1.0μg咖啡因/1ml色谱级甲醇Std.3:5.0μgcaffeine/1mlmethanolHPLCGrade标准3：5.0μg咖啡因/1ml色谱级甲醇Std.4:25.0μgcaffeine/1mlmethanolHPLCGrade标准4：25.0μg咖啡因/1ml色谱级甲醇Std.5:50.0μgcaffeine/1mlmethanolHPLCGrade标准5：50.0μg咖啡因/1ml色谱级甲醇Std.6:methanolHPLCGrade(blank)标准6：色谱级甲醇（空白） Std.5:weigh9.0to11.0mgcaffeineandfillupto200.0mlwithmethanolHPLC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:dilute50.0mlofStd.5to100.0mlwithmethanol标准4：取50.0ml标准5溶液用甲醇稀释至100.0mlStd.3:dilute10.0mlofStd.5to100.0mlwithmethanol标准3：取10.0ml标准溶液5用甲醇稀释至100.0mlStd.2:dilute20.0mlofStd.3to100.0mlwithmethanol标准2：取20.0ml标准3溶液用甲醇稀释至100.0mlStd.1:dilute10.0mlofStd.3to100.0mlwithmethanol标准1：取10.0ml标准3用甲醇稀释至100.0ml Settings:设置Column:RP-185um30-50x2,1-4.6mmorcapillary2000mmx0.12mmID色谱柱：RP-185um30-50x2,1-4.6mmorcapillary2000mmx0.12mmIDMobilephase:methanolHPLCGrade流动相：色谱级甲醇Oventemperature:40°C柱温箱温度：40°CFlowrate:1.0ml/min(adjustedbyusing100%methanol)流速：1.0ml/min（用100%甲醇调整）Detection:273nm检测波长：273nmInjectionvolume:20μl进样量：20ul Method方法:Injectionscheme:进样序列2xblank1xStd.11xStd.21xStd.31xStd.41xStd.52针空白，标准1-5依序各一针 Limits限度:r2≥0.999Remark:Asthistestemploysdifferenttestsolutionstobeinjected,itcoversalsothecheckofcorrectpositioningvialsintheautosampler.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 WAVELENGTHACCURACY波长准确度 Iftherearebuilt-intestproceduresforthedeterminationandadjustmentofwavelengthaccuracy,followtheinstructionsoftheinstrumentmanual.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 操作手册模板用友nc操作手册操作手册格式模板汽车起重机操作手册用友nc操作手册53 进行操作Inallothercasesusetheproceduredescribedbelow.在所有其它情况下采用以下程序Solutions:溶液DAD:caffeineStd.5fromthelinearitytesting线性测试中咖啡因标准溶液5号UV/VIS:1.0μg/mlanthracenesolutioninwater蒽水溶液 Settings:设置Mobilephase:15%acetonitrileinwater流动相：15%乙腈水溶液Column:RP18,5?m30-50x2,1-4.6mmorcapillary2.0mx0.12mmID色谱柱：RP18，5μm,30-50×2.1-4.6mm或毛细管柱20m×0.12mm内径Oventemperature:40°C柱箱温度：40℃Flowrate:1.0ml/min(adjustedbyusing15%acetonitrileinwater)流速：1.0ml/min（用15%的乙腈水溶液调节）Detection:scanfrom230nmto290nm(DAD)检测：自230至290nm扫描（DAD）Injectionvolume:20μl进样体积：20μl Method方法:DAD:Inject20μlofthecaffeinesolutionandrecordthespectrum.Themaximumisat272nmandtheminimumat244nm.DAD:咖啡因溶液进样20μl，记录色谱图，最大吸收波长为272nm，最小为244nm.UV/VIS:fillthecellwiththeanthracenesolutionandchangethewavelengthfrom248to254nmin1nmsteps;recordthemaximumofabsorption.Thetheoreticalvalueis251nm.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:≤2nm HPLCFLUORESCENCEDETECTOR荧光检测器 ThefollowingthreeparametersareproposedfortheperformanceoftheperiodicandmotivatedcheckoftheHPLCfluorescentdetector:下面三个参数建议作为荧光检测器性能周期自主检查 WAVELENGTHACCURACYEXCITATION波长激发准确度 Method:方法Rinseandfillthemeasuringcellwithde-ionizedwater用去离子水淋洗并充满检测池Adjusttheexcitationwavelengthto350nm.调节激发波长为350nmMeasuretheemissionandsubtract397nm(theoreticalvalue).测量发射波长减去397nm（理论值） Limits限度:±3nm WAVELENGTHACCURACYEMISSION波长发射准确度Method:方法Rinseandfillthemeasuringcellwithde-ionizedwater用去离子水淋洗并充满检测池Adjusttheemissionwavelengthto397nm.调节发射波长为397nmMeasuretheexcitationandsubtract350nm(theoreticalvalue).测量激发波长减去350nm（理论值） Limits限度:±3nm SENSITIVITY灵敏度Solutions:溶液QuinineHCl.2H2Osolutionconc.0.015μg/ml(=15ppb)二水盐酸喹啉溶液，浓度0.015μg/ml(=15ppb)Thequininesolutionispreparedwiththefollowingmobilephase:dissolve6.8gofpotassiumdihydrogenphosphateRand3.0gofhexylamineRin700mlofwaterR,adjusttopH2.8withdilutephosphoricacidR,add90mlofacetonitrileRanddiluteto1000.0mlwithwaterR.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ThechromatographicconditionsaresetaccordingtoPh.Eur.“QuinineHCl”(01/2005:0018),test“Othercinchonaalkaloids”,withmodifiedflowrateandacetonitrileconcentration.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。Mobilephase:asabove流动相：同上Column:RP18,5μm,250×4.6mm色谱柱:RP18,5μm,250×4.6mmFlowrate:1.2ml/min流速：1.2ml/minExcitationwavelength:350nm激发波长：350nmEmissionwavelength:397nm发射波长：397nmFlow-cellvolume:8μl(forthisexample,aWaters2475MultiFluorescentiedetectorwasused.Flow-cellvolumemayvarydependingontheinstrumentmanufacturer)流动池体积：8μl（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:Inject10μlofthequininesolutionandmeasurethepeakheight.进样喹啉溶液10μl，测量峰高度Inject10μloftheblankandmeasurethepeakheightofthenoise.进样空白溶液10μl，测量噪音峰高Dividethepeakheightofthequininesolutionby3timesthepeakheightofthenoise.将喹啉溶液峰高除以3倍噪音峰高Dividetheconcentrationofthequininesolutionbythepreviouslyobtainedfactor.将喹啉溶液浓度除以之前的系数Limits限度:≤0.5ppb HPLCELECTROCHEMICALDETECTOR高效液相电化学检测器 AccuracyandstabilityofthesignalaretheproposedparameterstobetestedduringtheperiodicandmotivatedcheckoftheHPLCelectrochemicaldetector.电化学检测器的信号准确度和稳定性是定期自主检测中需要测定的参数 ACCURACYANDSTABILITYOFTHESIGNAL信号准确度和稳定性 Settings:设定Cellpotentialofadummycell:800mV空白池电压：800mvRisetimefilter:0.1s增加时间过滤：0.1sRange:0.1nA范围：0.1nATemperature:30°C温度：30℃ Method方法:Accuracy:Measuretheelectriccurrentandsubtract2.67nA(theoreticalvalue)准确度：检测电流并减去2.67nA（理论值）Stability:Measurethenoiseoveraperiodof5minutes稳定性：连续5分钟测量噪音 Limits限度:Accuracy(cellcurrent):±0.05nA准确度（池电流）：±0.05nAStabilityofthesignal(Noise):max.2pAor20mV信号（噪音）稳定性：最大2pA或20mV HPLCRIDDETECTOR高效液相RID检测器SignaltoNoiseratioanddriftovertimearetheparametersproposedfortheperiodicandmotivatedcheckoftheHPLCRID(refractiveindex)detector.高效液相RID（折光率）检测器的信噪比和对时间的漂移是定期自主检测中需要测定的参数 SIGNALTONOISERATIO信噪比 Solutions溶液:Standardsolution:D-fructoseconcentratesolutionat4.0mg/ml(dilute200.0mgfructose+20mlwater+25.0mlacetonitrileupto50.0mlwithwaterforHPLC)标准溶液：D-左旋糖溶液4.0mg/ml（将200.0mg左旋糖+20ml水+25.0ml乙腈，用色谱纯水稀释至50.0ml） Settings设置:Column:spherisorbNH2(orequivalent)250×4.6mmorother色谱柱：spherisorbNH2(或相当的)250×4.6mm或其他Oventemperature:38°C柱温箱温度：38℃Flowrate:1.0ml/min流速:1.0ml/minInjectionvolume:20μl进样量:20μlMobilephase:0.253gsodiumhydrogenphosphateRin220ml+780mlacetonitrile流动相：0.253g磷酸氢二钠溶于220ml+780ml乙腈 Method方法:Afterequilibration,injectthreetimesablanksolutionofmobilephaseoveraruntimewherethesystemisstable.Measurethebaselinenoiseoveranappropriateperiod.平衡后，以流动相为空白进样三针，系统稳定运行适当时间，测定适当时长内噪音基线Thebaselinenoiseisacceptedifthemeanheightofthethreereplicatesis<1000μV.可接受基线噪音：三次平行进样平均值<1000μV Tocalculatethesignaltonoiseratio,injectthreetimesasolutionoffructoseat0.4mg/mlandcalculatethemeanofthethreereplicates.计算信噪比，左旋糖溶液0.4mg/ml进样三针，计算平均值 Limits限度:S/N≥10限度：信噪比≥10 DRIFTOVERADEFINEDPERIODOFTIME指定时长漂移 Method:方法Calculatetheslopeoftheamplitudeofrandomvariationsinthedetector’ssignalover1minute.计算任意一分钟信号随机波动幅度的斜率 Limits限度:±0.1mV/min限度：±0.1mV/minAlternatively,therequirementmaybeexpressedin△RI/minorin%offullscaleoftheselectedrange.也可以表达为△RI/min或者以选择量程的全范围%。 HPLCCDDETECTOR高效液相色谱CD检测器ThefollowingtestsareproposedtoperformtheperiodicandmotivatedcheckoftheHPLCCD(circulardichroism)detector.建议配备有CD（圆二图谱）检测器的HPLC进行以下周期和主动检查 LINEARITYANDSIGNALTONOISERATIO线性和信噪比 Solutions:溶液Referencesolution(a):dissolve25.0mgD(-)pantolactonein50.0mlwater对照溶液（a）：溶解25.0mgD（-）泛酰内酯于50.0ml水中Referencesolution(b):dilute2.0mlofreferencesolution(a)to10.0mlwithwater对照溶液（b）：取对照溶液（a）2.0ml用水稀释至10.0mlReferencesolution(c):dilute4.0mlofreferencesolution(a)to10.0mlwithwater对照溶液（c）：取对照溶液（a）4.0ml用水稀释至10.0ml.Referencesolution(d):dilute6.0mlofreferencesolution(a)to10.0mlwithwater对照溶液（d）：取对照溶液（a）6.0ml用水稀释至10.0ml.Referencesolution(e):dilute8.0mlofreferencesolution(a)to10.0mlwithwater对照溶液（e）：取对照溶液（a）8.0ml用水稀释至10.0ml.Referencesolution(f):dilute0.5mlofreferencesolution(b)to25.0mlwithwater对照溶液（f）：取对照溶液（b）0.5ml用水稀释至25.0ml. Settings:设定Column色谱柱:C18,150×4mm,5μmMobilephase流动相:acetonitrile乙腈:water水=10:90Flow流速:1.0ml/minDetection检测波长:225nmInjectionvolume进样量:20μl Method方法:CheckthelinearityofdeCD-andUV-signalofD(-)pantolactonereferencesolutiona,b,c,d,e.检查CD和UV对D（-）泛酰内酯对照溶液a,b,c,d,e信号的线性关系MeasurethenoiseoftheCD-signalofreferencesolution(f)between0–10min.检查CD对对照溶液（f）在1-10分钟的噪声-Calculatetheabsoluteconcentration(μg)inthecell 计算池中绝对浓度(μg)-Calculatethesignal-to-noiseratio(S/N)for0.01μginthecell 计算池中0.01μg信噪比（S/N）-CalculatethesensitivitywiththecalculatedS/NandthespecifiedS/N=2(0.01x2/S/Ncalculated)计算所得信噪比及信噪比为2(0.01x2/S/Ncalculated)时的灵敏度 Limits限度:Linearity:Thelinearityofthecalibrationlineobtainedwithreferencesolution(a,b,c,d,e)shouldber>0.999.线性：由对照液（a,b,c,d,e）所得的校正曲线线性系统应>0.999。Sensitivity:ThesensitivityatS/N=2shouldbebetterthan0.01μg.灵敏度：信噪比为2时灵敏度应好于0.01μg。S/Nratio:TheLimitforS/Nis>1.0andthesensitivityshouldbe0.020μgatmaximum.信噪比：信噪比为1.0时的限度和灵敏度最大为0.020μg。 DRIFTOVERADEFINEDPERIODOFTIME指定时间内飘移值 Settings设定:Column色谱柱:C18,150x4mm,5μmMobilephase流动相:acetonitrile乙腈:water水=10:90Flow流速:1.0ml/minDetection检测波长:290nmInjectionvolume进样量:20μlMethod方法:Injectwaterandstoptheflowafter5minutes.MeasuretheCD-signalfor1hour.进水样，停泵，5分钟后测量CD信号，持续1小时。Measurewiththecursorthedriftofthebaselinebetween5and65min.用标尺测量5到65分钟间的基线飘移Limits限度:Notmorethan0.1mdeg/h不超过0.1mdeg/h SPECTRACOMPARISON谱图比较 Solutions溶液:Referencesolution(a):dissolve5.0mgdexamethasonein10.0ml40%acetonitrile对照溶液（a）：溶解5.0mg地塞米松于10.0ml40%乙腈中。 Settings设定:Column色谱柱:C18,150x4mm,5μmMobilephase流动相:acetonitrile乙腈:water水=40:60Flow流速:1.0ml/minDetection检测波长:230nmInjectionvolume进样量:20ul Method方法:Comparethemaxima/minimaobtainedattheInstallationofthedetector(seetable).比较检测器安装结果（见下表）CDmax最大CDmin最小UVmax最大222nm22