EP80细菌内毒素检查法中英文对照教学文案

1、Good is good, but better carries it.精益求精，善益求善。EP80细菌内毒素检查法中英文对照-EP8.02.6.14细菌内毒素（中英文）2.6.14.BACTERIALENDOTOXINS细菌内毒素Thetestforbacterialendotoxins(BET)isusedtodetectorquantifyendotoxinsfromgram-negativebacteriausingamoebocytelysatefromthehorseshoecrab(LimuluspolyphenmusorTachypleustridentatus).Therea

2、re3techniquesforthistest:thegel-clottechniques,whichisbasedongelformation;theturbidimetrictechnique,basedonthedevelopmentofturbidityaftercleavageofanendogenousesubstrate;andthechromogenictechnique,basedonthedevelopmentofcolouraftercleavageofasyntheticpeptide-chromogencomplex.本法利用鲎试剂（从鲎美洲鲎或中国鲎变形细胞溶解物

3、制备而来）检测由革兰氏阴性菌产生的细菌内毒素或对内毒素进行定量。该检查包括三种方法：一为凝胶法，系利用鲎试剂与内毒素产生凝集反应的原理；第二种为浊度法（基于内源性底物断裂后，产生的浊度变化）；最后一种为显色法（得到的肽呈色基团复合物断裂后，检测反应混合物的色度）。Thefollowing6methodsaredescribedinthepresentchapter:这一章阐述了下面6种方法：MethodA.Gel-clotmethod:limittestMethodB.Gel-clotmethod:quantitativetestMethodC.Turbidimetrickineticmeth

4、odMethodD.ChromogenickineticmethodMethodE.Chromogenicend-pointmethodMethodF.Turbidimetricend-pointmethod方法A：凝胶法：限度试验方法B：凝胶法：定量试验方法C：动态浊度法方法D：动态显色法方法E：终点显色法方法F：终点浊度法Proceedbyanyofthe6methodsforthetest.Intheeventofdoubtordispute,thefinaldecisionismadebaseduponmethodAunlessotherwiseindicatedinthemonogr

5、aph.检测时，可用6种方法的任一种进行试验。当测定结果可疑或有争议时，除非另有规定，以专论中的方法A的测定结果为准。Thetestiscarriedoutinamannerthatavoidsendotoxincontamination.试验操作过程应防止内毒素的污染。1.APPARATUS仪器Depyrogenateallglasswareandotherheat-stableapparatusinahot-airovenusingavalidatedprocess.Acommonlyusedminimumtimeandtemperatureis30minutesat25?C.Ifempl

6、oyingplasticapparatus,suchasmicrotitreplatesandpipettetipsforautomaticpipetters,useapparatusshowntobefreeofdetectableendotoxinandwhichdoesnotinterfereinthetest.所有的玻璃器皿及由其他耐热材料制成的器皿需用已验证的工艺在热烘箱内进行去热原处理。去热原时，常用的最小时间和温度设置分别为30分钟和250。若使用塑料器械，如微孔板和微量进样器配套的吸头等，它们必须标明无内毒素并确对试验无干扰。NOTE:inthischapter,theterm

7、tubeincludesalltypesofreceptacles,forexamplemicrotitreplatewells.注：这一章中，“管”的意思包括其他任何反应容器，如微孔板中的孔。2.REAGENTS,TESTSOLUTIONS试剂、试液(1)Amoeboytelysate鲎试剂Amoebocytelysateisalyophilizedproductobtainedfromamoebocytelysatefromthehorseshoecrab(LimuluspolyphemusorTachypleustridentatus).Thisreagentrefersonlytoap

8、roductmanufacturedinaccordancewiththeregulationsofthecompetentauthority.鲎试剂物是从鲎（美洲鲎或中国鲎）的变形细胞产生的低压冻干产物。该试剂仅指在符合有关权威法规的生产条件下生产的鲎试剂产品。NOTE:amoebocytelysatereactswithsome-glucansinadditiontoendotoxins.Amoebocytelysatepreparationswhichdonotreactowithglucansareavailable;theyarepreparedbyremovingfromamoeb

9、ocytelysatetheGfactor,whichreactswithglucans,orbyinhibitingtheGfactorreactoingsystemofamoebocytelysate.Thesepreparationsmaybeusedforendotoxintestinginthepresenceofglucans.注：除了内毒素外，鲎试剂和-葡聚糖反应。也存在不与葡聚糖反应的鲎试剂；它们通过从变形细胞溶解物中除去G因子来制备，因为G因子会和葡聚糖反应，或者通过抑制变形细胞溶解物的G因子反应系统来制备。这些制剂可被用于葡聚糖存在情况下的内毒素测试。(2)Lysateso

10、lution鲎试液DissolveamoebocytelysateinwaterforBETorinabuffer,asrecommendedbythelysatemanufacturer,bygentlestirring.Storethereconstitutedlysate,refrigeratedorfrozen,asindicatedbythemanufacturer.通过缓慢搅拌，将鲎试剂溶于水或溶于厂家推荐的缓冲液中来进行细菌内毒素试验（BET）。按照厂商的指示，冷藏或冷冻储存重新鲎试液。(3)WaterforBET(waterforbacterialendotoxinstest)

11、BET用水WaterforinjectionsRorwaterproducedbyotherproceduresthatshowsnoreactionwiththelysateemployedatthedetectionlimitofthereagent.BET用水指注射用水R或由其他方法制取的内毒素含量低于所用鲎试剂的检测限的水。3.PREPARATIONOFTHESTANDARDENDOTOXINSTOCKSOLUTION（内毒素储备标准溶液的制备）Thestandardendotoxinstocksolutionispreparedfromanendotoxinreferencestan

12、dardthathasbeencalibratedagainsttheInternationalStandard,forexampleendotoxinstandardBRP.用内毒素标准品制备内毒素储备标准溶液；所用的内毒素标准品必须先用国际标准品校准，如内毒素标准BRP。EndotoxinisexpressedinInternationalUnits(IU).TheequivalenceinIUoftheInternationalStandardisstatedbyWorldHealthOrganisation.内毒素以国际单位（IU）表示。IU的换算见国际卫生组织（WHO）公布的国际标准

13、。NOTE:oneInternationalUnit(IU)ofendotoxinisequaltooneEndotoxinUnit(E.U.).注：一国际单位（IU）内毒素相当于一个内毒素单位（E.U.）。Followthespecificationsinthepackageleafletandonthelableforpreparationandstorageofthestandardendotoxinstocksolution.根据包装说明书上的标准和内毒素储备标准溶液的标签上关于制备和贮存的说明。4.PREPARATIONOFTHESTANDARDENDOTOXINSOLUTIONS（

14、内毒素标准溶液的制备）Aftervigorouslymixingthestandardendotoxinstocksolution,prepareappropriateserialdilutionsofthissolutionusingwaterforBET.充分混合内毒素储备标准溶液后，用细菌内毒素试验检查用水（BET用水）稀释，制成适当的系列稀释液，即得BET用内毒素标准溶液。Usethesolutionassoonaspossibletoavoidlossofactivitybyadsorption.得到的稀释液应尽快使用，以免因吸附而导致活性损失。5.PREPARATIONOFTHET

15、ESTSOLUTIONS供试品溶液的制备PreparethetestsolutionsbydissolvingordilutingactivesubstancesormedicinalproductsusingwaterforBET.Somesubstancesorpreparationsmaybemoreappropriatelydissolvedordilutedinotheraqueoussolutions.Ifnecessary,adjustthepHofthetestsolution(ordilutionthereof)sothatthepHofthemixtureofthelysa

16、teandtestsolutionfallswithinthepHrangespecifiedbythelysatemanufacturer,usually6.0to8.0.thepHmaybeadjustedbytheuseofacid,baseorasuitablebuffer,asrecommendedbythelysatemanufacturer.Acidsandbasesmaybepreparedfromconcentratesorsol8idswithwaterforBETincontainersfreeofdetectableendotoxin.Buffersmustbevali

17、datedtobefreeofdetectableendotoxinandinterferingfactors.除非另有说明，以BET用水溶解或稀释活性成分或药品来制备供试品溶液。某些成分或药品可能在其它的水溶液中溶解度更高。如有必要，调节待测溶液（或它的稀释液）的pH值，使鲎试剂和供试品溶液的混合物的pH值在所选鲎试剂生产商的指定pH范围内。一般要求供试品溶液的pH值范围为6.08.0。可用鲎试剂生产商推荐的酸、碱或适当的缓冲溶液来调解pH值。酸或碱溶液须用BET检查用水在去除内毒素的容器中配制。缓冲液必须经过验证不含内毒素和干扰因子。DETERMINATIONOFTHEMAXIMUMVAL

18、IDDILUTION确定最大有效稀释倍数TheMaximumValidDilution(MVD)isthemaximumallowabledilutionofasampleatwhichtheendotoxinlimitcanbedetermined.DeterminetheMVDusingthefollowingformulae:最大有效稀释倍数（MVD）指可检测出内毒素限值的供试品溶液的最大稀释倍数。MVD按下列公式确定：MVD=EndotoxinlimitconcentrationoftestsolutionMVD=内毒素限值供试品溶液浓度/Endotoxinlimit:theendot

19、oxinlimitforactivesubstancesadministeredparenterally,definedonthebasisofdose,isequaltoKM内毒素限值：在剂量的基础上，注射药物的活性成分的内毒素限度为K/MK=thresholdpyrogenicdoseofendotoxinperkilogramofbodymass,M=maximumrecommendedbolusdoesofproductperkilogramofbodymass.K=人每公斤体重每小时最大可接受的内毒素剂量（EU/kg）M人用每公斤体重每小时的最大供试品剂量。Whentheproduc

20、tistoeinjectedatfrequentintervalsorinfusedcontinuously,Misthemaximumtotaldoseadministeredinasinglehourperiod.Theendotoxinlimitforactivesubstancesadministeredparenterallyisspecifiedinunitssuchas:IU/mL,IU/mg,IU/Unitofbiologicalactivity,ect,inmonographs.专论中，注射剂活性成分的内毒素限度的单位有IU/ml、IU/mg、IU/生物活性单位等。Conce

21、ntrationoftestsolution:供试品溶液的浓度表示法：mg/mLiftheendotoxinlimitisspecifiedbymass(IU/mg),当内毒素限度以质量（IU/mg）表示时，用mg/ml表示浓度。Units/mLiftheendotoxinlimitisspecifiedbyunitofbiologicalactivity(IU/Unit),当内毒素限度以（IU/ml）表示时，用ml/ml表示浓度。ml/mLiftheendotoxinlimitisspecifiedbyvolume(IU/mL).当内毒素限度以生物单位（IU/Unit）表示时，用Units/

22、ml表示浓度。=thelablelledlysatesensitivityinthegel-clottechniques(IU/mL)orthelowestconcentrationusedinthestandardcurveoftheturbidimetricorchromogenictechniques.=指凝胶法中鲎试剂的标示灵敏度，或指浊度法或显色法使用的标准回归曲线上最低点（IU/ml)的对应值。7.GEL-CLOTTECHNIQUE(METHODSAANDB)凝胶法（方法A和B）Thegel-clottechniquesallowdetectionorquantificationo

23、fendotoxinsandisbasedonclottingofthelysateinthepresenceofendotoxins.Theminimumconcentrationofendotoxinsrequiredtocausethelysatetoclotunderstandardconditionsisthelabeledlysatesensitivity.Toensureboththeprecisionandvalidityofthetest,confirmthelabeledlysatesensitivityandperformthetestforinterferingfact

24、orsasdescribedunder1.Preparatorytesting.凝胶法系通过鲎试剂与内毒素产生凝集反应的原理来检测或定量内毒素的方法。在标准条件下，可引起鲎试液凝集的内毒素的最低浓度即为鲎试剂的标示灵敏度。为确保试验结果精确、有效，应根据预备试验中的说明开展试验，复核鲎试剂的标示灵敏度和试验的干扰因素。1.PREPARATORYTESTING预备试验(i)Confirmationofthelabeledlysatesensitivity鲎试剂的标示灵敏度复核试验Confirmin4replicatesthelabeledsensitivity,expressedinIU/mL,

25、ofthelysatesolutionpriortouseinthetest.Confirmationofthelysatesensitivityiscarriedoutwhenanewlotoflysateisusedorwhenthereisanychangeinthetestconditionswhichmayaffecttheoutcomeofthetest.灵敏度用IU/ml表示。应在鲎试剂用于检查内毒素之前对灵敏度进行复核；复核试验需要制备标示灵敏度的4支平行管。当使用新批号的鲎试剂或试验条件发生了任何可能影响检验结果的改变时，应进行鲎试剂灵敏度复核试验。Preparestanda

26、rdsolutionsofatleast4concentrationsequivalentto2,0.5and0.25bydilutingthestandardendotoxinstocksolutionwithwaterforBET.用BET用水稀释内毒素储备标准溶液，制成2、0.5和0.25四种浓度的标准溶液。Mixavolumeofthelysatesolutionwithanequalvolumeof1ofthestandardsolutions(suchas0.1mLaliquots)ineachtube.Whensingletestvialsorampoulescontaining

27、lyophilizedlysateareemployed,addsolutionsofstandardsdirectlytothevialorampoule.Incubatethereactionmixtureforaconstantperiodaccordingtotherecommendationsofthelysatemanufacturer(usuallyat371?Cfor602min),avoidingvibration.Testtheintegrityofthegel:fortubes,takeeachtubeinturndirectlyfromtheincubatorandin

28、vertitthroughapproximately180?inonesmoothmotion.Ifafirmgelhasformedthatremainsinplaceuponinversion,recordtheresultaspositive.Aresultisnegativeifanintactgelisnotformed.在每支试管中，分别混合等体积（通常为0.1mL)的鲎试液和标准溶液。如使用的是装有鲎试剂冻干粉末的西林瓶或安瓿，可向其中直接加入溶液。按照鲎试剂生产商的建议，将反应混合物孵育一段时间(通常在371下保温602min)，在此过程中，不能振动试管。凝胶的完整性测试：如使

29、用试管作为容器，先将试管从保温箱中取出，再缓缓将试管倒转180o。如果形成了坚实的凝胶，倒转后凝胶不移位，此时将该项检查的结果记录为阳性。如未能形成坚实且不变形的凝胶，该项检查的结果即为阴性。Thetestisconsideredvalidwhenthelowestconcentrationofthestandardsolutionsshowsanegativeresultinallreplicatetests.Theend-pointisthelowestconcentrationintheseriesofdecreasingconcentrationsofstandardendotoxin

30、thatclotsthelystae.Determinethegeometricmeanend-pointconcentrationbycalculatingthemeanofthelogarithmsoftheend-pointconcentrationsofthe4dilutionseries,taketheantilogarithmofthisvalue,asindicatedbythefollowingexpression:仅在最低浓度的标准溶液的所有平行管的检查结果均为阴性的情况下，试验方为有效。反应终点浓度指系列递减的内毒素浓度中最后一个呈阳性结果的浓度。将终点浓度取对数，计算它们

31、的平均值，再将平均值的结果取反对数，最后的表达式如下：Geometricmeanend-pointconcentration=antilogefe=sumofthelogend-pointconcentrationsofthedilutionseriesused,f=numberofreplicates.终点浓度的几何平均值log-1(e/f)e所用系列溶液的终点浓度的对数值的和f平行管的数量Thegeometricmeanend-pointconcentrationisthemeasuredsensitivityofthelysatesolution(IU/mL).Ifthisisnotle

32、ssthan0.5andnotmorethan2,thelabeledsensitivityisconfirmedandisusedinthetestperformedwiththislysate.反应终点的浓度的几何平均值即为鲎试剂灵敏度的测量值（IU/ml）。当终点浓度的几何平均值在0.5至2.0之间时，可判定受试鲎试剂的标示灵敏度为，可用于内毒素的检查。(ii)Testforinterferingfactors干扰因素试验PreparesolutionsA,B,CandDasshowninTable2.6.14-1,andusethetestsolutionsatadilutionles

33、sthantheMVD,notcontaininganydetectableendotoxins,operatingasdescribedunder1.preparatorytesting,(i)Confirmationofthelabeledlysatesensitivity.按表2.6.14.-1制备溶液A、B、C、D。供试品的稀释度不得超过MVD，且供试品溶液不能检查出内毒素，具体操作见（1）预备试验，（i）鲎试剂标示灵敏度的复核试验项。Thegeometricmeanend-pointconcentrationsofsolutionsBandCaredeterminedusingthe

34、expressiondescribedin1.Preparatorytesting,(i)Confirmationofthelabeledlysatesensitivity.根据（1）预备试验下的（i）鲎试剂标示灵敏度的复核试验项中列出的公式，计算B和C溶液的终点浓度的几何平均值。Table2.6.14.-1SolutionEndotoxinconcentration/solutiontowhichendotoxinisaddedDiluentDilutionfactorEndotoxinconcentrationNumberofreplicatesANone/Testsolution-4B2

35、/TestsolutionTestsolution1248210.50.254444C2/WaterforBETWaterforBET1248210.50.252222DNone/WaterforBET-2SolutionA=solutionofthepreparationbeingexaminedthatisfreeofdetectableendotoxins.SolutionB=testforinterference.SolutionC=controlofthelabeledlysatesensitivity.SolutionD=negativecontrol(waterforBET).表

36、2.6.14-1溶液内毒素浓度/配制内毒素的溶液稀释剂稀释倍数稀释后内毒素的浓度平行管数A无/供试品溶液4B2/供试品溶液供试品溶液1248210.50.254444C2/BET用水BET用水1248210.50.252222D0/BET用水2A=经检查无内毒素的溶液B=干扰实验用C=鲎试剂标示灵敏度的对照品D=阴性对照品（BET检查用水）Thetestforinterferingfactorsmustberepeatedwhenanychangesaremadetotheexperimentalconditionsthatarelikelytoinfluencetheresultofthet

37、est.如试验条件发生了任何可能影响到试验结果的变化，须重新进行干扰因素试验。ThetestisconsideredvalidwhenallreplicatesofsolutionsAandDshownoreactionandtheresultofsolutionCconfirmsthelabeledlysatesensitivity.只有当溶液A和D的所有平行管中无反应、且溶液C的结果在复核的鲎试剂灵敏度范围内时，试验方有效。IfthesensitivityofthelysatedeterminedwithsolutionBisnotlessthan0.5andnotgreaterthan2

38、,thetestsolutiondoesnotcontaininterferingfactorsundertheexperimentalconditionsused.Otherwise,thetestsolutioninterfereswiththetest.经测定，如果溶液B的灵敏度在0.5，2.0间，可判定供试品溶液在该实验条件下，对实验无干扰；反之则判定供试品溶液对试验能形成干扰。IfthepreparationbeingexaminedinterfereswiththetestatadilutionlessthantheMVD,repeatthetestforinterferingfa

39、ctorsusingagreaterdilution,notexceedingtheMVD.Theuseofamoresensitivelysatepermitsagreaterdilutionofthepreparationbeingexaminedandthismaycontributetotheeliminationofinterference.若供试品溶液在小于MVD的稀释倍数下对试验有干扰，应将供试品溶液进行不超过MVD的进一步稀释，再重复干扰试验。使用灵敏度更高的鲎试剂进行内毒素的检查时，因为更高灵敏度鲎试剂能排除实验的干扰因素，供试品的稀释倍数也可适当放宽。Interferenc

40、emaybeovercomebysuitablevalidatedtreatment,suchasfiltration,neutralization,dialysisorheattreatment.Toestablishthatthetreatmentchoseeffectivelyeliminatesinterferencewithoutlossofendotoxins,repeatthetestforinterferingfactorsusingthepreparationbeingexaminedtowhichthestandardendotoxinhasbeenaddedandwhic

41、hhasthenbeensubmittedtothechosentreatment.可通过其他适宜的方法（如过滤、中和、透析或加热处理等）排除干扰。为确保所选择的处理方法能有效地排除干扰且不会使内毒素失去活性，要使用预先添加了标准内毒素再经过处理的供试品溶液进行干扰实验。2.LIMITTEST(METHODA)限度试验（方法A）(i)Procedure方法PreparesolutionsA,B,CandDasshowninTable2.6.14.-2,andperformthetestonthesesolutionsfollowingtheproceduredescribedunder1.Pr

42、eparatorytesting,(i)Confirmationofthelabeledlysatesensitivity.按表2.6.14-2制备溶液A、B、C、D。实验方法见（1）预备试验的（i）鲎试剂标示灵敏度的复核试验项。Table2.6.14.-2SolutionEndotoxinconcentration/SolutiontowhichendotoxinisaddedNumberofreplicatesANone/Dilutedtestsolution2B2/Dilutedtestsolution2C2/WaterforBET2DNone/WaterforBET2表2.6.14-2

43、溶液内毒素浓度/配制内毒素的溶液平行管数A0/供试品溶液2B2/供试品溶液2C2/BET用水2D0/BET用水2PreparesolutionAandsolutionB(positiveproductcontrol)usingadilutionnotgreaterthantheMVDandtreatmentsasdescribedin1.Preparatorytesting,(ii)Testforinterferingfactors.SolutionsBandC(positivecontrols)containthestandardendotoxinataconcentrationcorres

44、pondingtotwicethelabeledlysatesensitivity.SolutionD(negativecontrol)consistsofwaterforBET.制备溶液A、B（供试品阳性对照），稀释倍数不得超过MVD。按照（1）预备实验，（ii）干扰因素实验项下的说明开展实验。溶液B、C（阳性对照）所含标准内毒素的浓度为鲎试剂标示灵敏度的两倍。溶液D（阴性对照）为BET用水。(ii)Interpretation结果判断ThetestisconsideredvalidwhenbothreplicatesofsolutionBandCarepositiveandthoseofs

45、olutionDarenegative.只有溶液B和C的平行管的试验结果均为阳性、溶液D的平行管的试验结果均为阴性时，试验方有效。WhenanegativeresultisfoundforbothreplicatesofsolutionA,thepreparationbeingexaminedcomplieswiththetest.溶液A的平行管的试验结果均为阴性时，可判定供试品符合试验的内毒素限度要求。WhenapositiveresultisfoundforbothreplicatesofsolutionA,thepreparationbeingexamineddoesnotcomplyw

46、iththetest.溶液A的平行管的检查结果均为阳性时，可判定供试品不符合试验的内毒素限度要求。WhenapositieresultisfoundforonereplicateofsolutionAandanegativeresultisfoundfortheothers,repeatthetest.当溶液A的平行管其中一个检查结果为阳性，另一个为阴性时，重复试验。Intherepeattest,thepreparationbeingexaminedcomplieswiththetestifanegativeresultisfoundforbothreplicatesofsolutionA.

47、ThepreparationdoesnotcomplywiththetestifapositiveresultisfoundforoneorbothreplicatesofsolutionA.在重复试验中，溶液A的平行管的试验结果均为阴性时，可判定供试品符合试验的内毒素限度要求。溶液A的平行管的检查结果有一个或均为阳性时，可判定供试品不符合试验的内毒素限度要求。However,ifthepreparationdoesnotcomplywiththetestatadilutionlessthantheMVD,thetestmayberepeatedusingagreaterdilution,no

48、texceedingtheMVD.但是，如果供试品不符合规定是在供试品的稀释倍数小于MVD的情况下，应将供试品溶液稀释至较大但不超过MVD的倍数，再次开展试验。3.QUANTITATIVETEST(METHODB)(i)ProcedureThetestquantifiesbacterialendotoxinsinthetestsolutionbytitrationtoanend-point.PreparesolutionsA,B,CandDasshowninTable2.6.14.-3,andtestthesesolutionsaccordingtotheproceduredescribedu

49、nder1.Preparatorytesting,(i)Confirmationofthelabelledlysatesensitivity.通过将供试品滴定至反应终点，对供试品所含的细菌内毒素定量。按表2.6.14-3制备溶液A、B、C、D。具体实验操作见（1）预备试验的（i）鲎试剂标示灵敏度的复核试验项。Table2.6.14-3SolutionEndotoxinConcentration/SolutiontowhichEndotoxinisAddedDiluentDilutionFactorInitialEndotoxinConcentrationNumberofReplicatesAH

50、YPERLINK:29240/uspnf/pub/data/v29240/usp29nf24s0_c85.xmllusp29nf24s0_fc8510anone/samplesolutionWaterforBET12482222BHYPERLINK:29240/uspnf/pub/data/v29240/usp29nf24s0_c85.xmllusp29nf24s0_fc8511b2/samplesolution122CHYPERLINK:29240/uspnf/pub/data/v29240/usp29nf24s0_c85.xmllusp29nf24s0_fc8512c2/WaterforB

51、ETWaterforBET1248210.50.252222DHYPERLINK:29240/uspnf/pub/data/v29240/usp29nf24s0_c85.xmllusp29nf24s0_fc8513dnone/WaterforBET2SolutionA=testsolutionatthedilution,notexceedingtheMVD,withwhichthetestforinterferingfactorswascarriedout.SubsequentdilutionofthetestsolutionmustnotexceedtheMVD.UsewaterforBET

52、tomakeadilutionseriesof4tubescontainingthetestsolutionatconcentrationsof1,1/2,1/4and1/8,relativetothedilutionusedinthetestforinterferingfactors.OtherdilutionsuptotheMVDmaybeusedasappropriate.SolutionB=solutionAcontainingstandardendotoxinataconcentrationof2(positiveproductcontrol).SolutionC=adilution

53、seriesof4tubesofwaterforBETcontainingthestandardendotoxinatconcentrationsof2,0.5and0.25.SolutionD=waterforBET(negativecontrol).表2.6.14-3溶液内毒素浓度/配制内毒素的溶液稀释剂稀释倍数稀释后内毒素的浓度平行管数A无/供试品溶液BET用水1248-2222B2/供试品溶液122C2/BET用水BET用水1248210.50.252222D无/BET用水2溶液A=不超过MVD并且通过干扰试验的供试品溶液。从通过干扰试验的稀释倍数开始，用BET用水稀释至1倍、2倍、4

54、倍和8倍，每一浓度准备两份稀释液，最后的稀释倍数不得超过MVD，并且确定稀释液通过了干扰试验。也可以准备其它合适倍数的稀释液。溶液B=2浓度标准内毒素的溶液A（供试品阳性对照）溶液C=含有2、0.5和0.25浓度标准内毒素的BET用水系列，每一浓度准备两份。溶液D=BET用水（阴性对照）(ii)CalculationandinterpretationThetestisconsideredvalidwhenthefollowing3conditionsaremet:(a)bothreplicatesofsolutionD(negativecontrol)arenegative,(b)bothre

55、plicatesofsolutionB(positiveproductcontrol)arepositive,(c)thegeometricmeanend-pointconcentrationofsolutionCisintherangeof0.5to2.(ii)计算和结果判断符合下列三个条件时，可将试验判为有效：溶液D（阴性对照）的两组平行管均显阴性，（b）溶液B（供试品阳性对照）的两个平行管均显阳性，（c）溶液C的终点浓度的几何平均值在0.52之间。TodeterminetheendotoxinconcentrationofsolutionA,calculatetheend-pointco

56、ncentrationforeachreplicate,bymultiplyingeachend-pointdilutionfactorby.Theendotoxinconcentrationinthetestsolutionistheend-pointconcentrationofthereplicates.Ifthetestisconductedwithadilutedtestsolution,calculatetheconcentrationofendotoxinintheoriginalsolutionbymultiplyingtheresultbythedilutionfactor.

57、Ifnoneofthedilutionsofthetestsolutionispositiveinavalidtest,reporttheendotoxinconcentrationaslessthan(or,ifadilutedsamplewastested,reportaslessthanthelowestdilutionfactorofthesample).Ifalldilutionsarepositive,theendotoxinconcentrationisreportedasequaltoorgreaterthanthelargestdilutionfactormultiplied

58、by(e.g.inTable2.6.14.-3,theinitialdilutionfactor8).Thepreparationbeingexaminedmeetstherequirementsofthetestiftheendotoxinconcentrationinbothreplicatesislessthanthatspecifiedinthemonograph.测定溶液A的内毒素浓度，计算溶液A的系列稀释液的终点浓度时，应将内毒素浓度记为终点稀释倍数乘以。供试品的内毒素浓度指这些平行管的反应终点浓度的几何平均值。如试验使用的是供试品的稀释液，计算时，将检查结果乘以稀释倍数，即得到原

59、始溶液的内毒素浓度。如在有效的试验中，供试品的检查结果均显阴性，应记为内毒素浓度小于（如实验使用的是供试品的稀释液，检查结果应记为内毒素浓度小于乘以供试品的最小稀释倍数）。如供试品的所有结果均为阳性，应记为内毒素浓度大于或等于最大的稀释倍数乘以（如，在表2.6.14-3中，初始稀释倍数8）。如供试品内毒素的浓度小于其专论中的规定浓度，可判定供试品符合检查要求。ETRICQUANTITATIVETECHNIQUES(METHODSC,D,EANDF)8.光度测定法（方法C、D、E和F）（1）.TURBIDIMETRICTECHNIQUE(METHODSCANDF)（1）浊度法（方法C和F）Thi

60、stechniqueisaphotometrictesttomeasuretheincreaseinturbidity.Basedonthetestprincipleemployed,thistechniquemaybeclassifiedasbeingeithertheend-point-turbidimetrictestorthekinetic-turbidimetrictest.本法是利用光学试验来测定浊度增加的方法。根据检测原理，这种方法又可分为终点浊度法和动态浊度法。Theend-point-turbidimetrictest(MethodF)isbasedonthequantita