EP80細(xì)菌內(nèi)毒素檢查法中英文對(duì)照教學(xué)文案

1、Good is good, but better carries it.精益求精，善益求善。EP80細(xì)菌內(nèi)毒素檢查法中英文對(duì)照-EP8.02.6.14細(xì)菌內(nèi)毒素（中英文）2.6.14.BACTERIALENDOTOXINS細(xì)菌內(nèi)毒素Thetestforbacterialendotoxins(BET)isusedtodetectorquantifyendotoxinsfromgram-negativebacteriausingamoebocytelysatefromthehorseshoecrab(LimuluspolyphenmusorTachypleustridentatus).Therea

2、re3techniquesforthistest:thegel-clottechniques,whichisbasedongelformation;theturbidimetrictechnique,basedonthedevelopmentofturbidityaftercleavageofanendogenousesubstrate;andthechromogenictechnique,basedonthedevelopmentofcolouraftercleavageofasyntheticpeptide-chromogencomplex.本法利用鱟試劑（從鱟美洲鱟或中國鱟變形細(xì)胞溶解物

3、制備而來）檢測(cè)由革蘭氏陰性菌產(chǎn)生的細(xì)菌內(nèi)毒素或?qū)?nèi)毒素進(jìn)行定量。該檢查包括三種方法：一為凝膠法，系利用鱟試劑與內(nèi)毒素產(chǎn)生凝集反應(yīng)的原理；第二種為濁度法（基于內(nèi)源性底物斷裂后，產(chǎn)生的濁度變化）；最后一種為顯色法（得到的肽呈色基團(tuán)復(fù)合物斷裂后，檢測(cè)反應(yīng)混合物的色度）。Thefollowing6methodsaredescribedinthepresentchapter:這一章闡述了下面6種方法：MethodA.Gel-clotmethod:limittestMethodB.Gel-clotmethod:quantitativetestMethodC.Turbidimetrickineticmeth

4、odMethodD.ChromogenickineticmethodMethodE.Chromogenicend-pointmethodMethodF.Turbidimetricend-pointmethod方法A：凝膠法：限度試驗(yàn)方法B：凝膠法：定量試驗(yàn)方法C：動(dòng)態(tài)濁度法方法D：動(dòng)態(tài)顯色法方法E：終點(diǎn)顯色法方法F：終點(diǎn)濁度法Proceedbyanyofthe6methodsforthetest.Intheeventofdoubtordispute,thefinaldecisionismadebaseduponmethodAunlessotherwiseindicatedinthemonogr

5、aph.檢測(cè)時(shí)，可用6種方法的任一種進(jìn)行試驗(yàn)。當(dāng)測(cè)定結(jié)果可疑或有爭議時(shí)，除非另有規(guī)定，以專論中的方法A的測(cè)定結(jié)果為準(zhǔn)。Thetestiscarriedoutinamannerthatavoidsendotoxincontamination.試驗(yàn)操作過程應(yīng)防止內(nèi)毒素的污染。1.APPARATUS儀器Depyrogenateallglasswareandotherheat-stableapparatusinahot-airovenusingavalidatedprocess.Acommonlyusedminimumtimeandtemperatureis30minutesat25?C.Ifempl

6、oyingplasticapparatus,suchasmicrotitreplatesandpipettetipsforautomaticpipetters,useapparatusshowntobefreeofdetectableendotoxinandwhichdoesnotinterfereinthetest.所有的玻璃器皿及由其他耐熱材料制成的器皿需用已驗(yàn)證的工藝在熱烘箱內(nèi)進(jìn)行去熱原處理。去熱原時(shí)，常用的最小時(shí)間和溫度設(shè)置分別為30分鐘和250。若使用塑料器械，如微孔板和微量進(jìn)樣器配套的吸頭等，它們必須標(biāo)明無內(nèi)毒素并確對(duì)試驗(yàn)無干擾。NOTE:inthischapter,theterm

7、tubeincludesalltypesofreceptacles,forexamplemicrotitreplatewells.注：這一章中，“管”的意思包括其他任何反應(yīng)容器，如微孔板中的孔。2.REAGENTS,TESTSOLUTIONS試劑、試液(1)Amoeboytelysate鱟試劑Amoebocytelysateisalyophilizedproductobtainedfromamoebocytelysatefromthehorseshoecrab(LimuluspolyphemusorTachypleustridentatus).Thisreagentrefersonlytoap

8、roductmanufacturedinaccordancewiththeregulationsofthecompetentauthority.鱟試劑物是從鱟（美洲鱟或中國鱟）的變形細(xì)胞產(chǎn)生的低壓凍干產(chǎn)物。該試劑僅指在符合有關(guān)權(quán)威法規(guī)的生產(chǎn)條件下生產(chǎn)的鱟試劑產(chǎn)品。NOTE:amoebocytelysatereactswithsome-glucansinadditiontoendotoxins.Amoebocytelysatepreparationswhichdonotreactowithglucansareavailable;theyarepreparedbyremovingfromamoeb

9、ocytelysatetheGfactor,whichreactswithglucans,orbyinhibitingtheGfactorreactoingsystemofamoebocytelysate.Thesepreparationsmaybeusedforendotoxintestinginthepresenceofglucans.注：除了內(nèi)毒素外，鱟試劑和-葡聚糖反應(yīng)。也存在不與葡聚糖反應(yīng)的鱟試劑；它們通過從變形細(xì)胞溶解物中除去G因子來制備，因?yàn)镚因子會(huì)和葡聚糖反應(yīng)，或者通過抑制變形細(xì)胞溶解物的G因子反應(yīng)系統(tǒng)來制備。這些制劑可被用于葡聚糖存在情況下的內(nèi)毒素測(cè)試。(2)Lysateso

10、lution鱟試液DissolveamoebocytelysateinwaterforBETorinabuffer,asrecommendedbythelysatemanufacturer,bygentlestirring.Storethereconstitutedlysate,refrigeratedorfrozen,asindicatedbythemanufacturer.通過緩慢攪拌，將鱟試劑溶于水或溶于廠家推薦的緩沖液中來進(jìn)行細(xì)菌內(nèi)毒素試驗(yàn)（BET）。按照廠商的指示，冷藏或冷凍儲(chǔ)存重新鱟試液。(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內(nèi)毒素含量低于所用鱟試劑的檢測(cè)限的水。3.PREPARATIONOFTHESTANDARDENDOTOXINSTOCKSOLUTION（內(nèi)毒素儲(chǔ)備標(biāo)準(zhǔn)溶液的制備）Thestandardendotoxinstocksolutionispreparedfromanendotoxinreferencestan

12、dardthathasbeencalibratedagainsttheInternationalStandard,forexampleendotoxinstandardBRP.用內(nèi)毒素標(biāo)準(zhǔn)品制備內(nèi)毒素儲(chǔ)備標(biāo)準(zhǔn)溶液；所用的內(nèi)毒素標(biāo)準(zhǔn)品必須先用國際標(biāo)準(zhǔn)品校準(zhǔn)，如內(nèi)毒素標(biāo)準(zhǔn)BRP。EndotoxinisexpressedinInternationalUnits(IU).TheequivalenceinIUoftheInternationalStandardisstatedbyWorldHealthOrganisation.內(nèi)毒素以國際單位（IU）表示。IU的換算見國際衛(wèi)生組織（WHO）公布的國際標(biāo)準(zhǔn)

13、。NOTE:oneInternationalUnit(IU)ofendotoxinisequaltooneEndotoxinUnit(E.U.).注：一國際單位（IU）內(nèi)毒素相當(dāng)于一個(gè)內(nèi)毒素單位（E.U.）。Followthespecificationsinthepackageleafletandonthelableforpreparationandstorageofthestandardendotoxinstocksolution.根據(jù)包裝說明書上的標(biāo)準(zhǔn)和內(nèi)毒素儲(chǔ)備標(biāo)準(zhǔn)溶液的標(biāo)簽上關(guān)于制備和貯存的說明。4.PREPARATIONOFTHESTANDARDENDOTOXINSOLUTIONS（

14、內(nèi)毒素標(biāo)準(zhǔn)溶液的制備）Aftervigorouslymixingthestandardendotoxinstocksolution,prepareappropriateserialdilutionsofthissolutionusingwaterforBET.充分混合內(nèi)毒素儲(chǔ)備標(biāo)準(zhǔn)溶液后，用細(xì)菌內(nèi)毒素試驗(yàn)檢查用水（BET用水）稀釋，制成適當(dāng)?shù)南盗邢♂屢?，即得BET用內(nèi)毒素標(biāo)準(zhǔn)溶液。Usethesolutionassoonaspossibletoavoidlossofactivitybyadsorption.得到的稀釋液應(yīng)盡快使用，以免因吸附而導(dǎo)致活性損失。5.PREPARATIONOFTHET

15、ESTSOLUTIONS供試品溶液的制備PreparethetestsolutionsbydissolvingordilutingactivesubstancesormedicinalproductsusingwaterforBET.Somesubstancesorpreparationsmaybemoreappropriatelydissolvedordilutedinotheraqueoussolutions.Ifnecessary,adjustthepHofthetestsolution(ordilutionthereof)sothatthepHofthemixtureofthelysa

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

17、datedtobefreeofdetectableendotoxinandinterferingfactors.除非另有說明，以BET用水溶解或稀釋活性成分或藥品來制備供試品溶液。某些成分或藥品可能在其它的水溶液中溶解度更高。如有必要，調(diào)節(jié)待測(cè)溶液（或它的稀釋液）的pH值，使鱟試劑和供試品溶液的混合物的pH值在所選鱟試劑生產(chǎn)商的指定pH范圍內(nèi)。一般要求供試品溶液的pH值范圍為6.08.0?？捎明c試劑生產(chǎn)商推薦的酸、堿或適當(dāng)?shù)木彌_溶液來調(diào)解pH值。酸或堿溶液須用BET檢查用水在去除內(nèi)毒素的容器中配制。緩沖液必須經(jīng)過驗(yàn)證不含內(nèi)毒素和干擾因子。DETERMINATIONOFTHEMAXIMUMVAL

18、IDDILUTION確定最大有效稀釋倍數(shù)TheMaximumValidDilution(MVD)isthemaximumallowabledilutionofasampleatwhichtheendotoxinlimitcanbedetermined.DeterminetheMVDusingthefollowingformulae:最大有效稀釋倍數(shù)（MVD）指可檢測(cè)出內(nèi)毒素限值的供試品溶液的最大稀釋倍數(shù)。MVD按下列公式確定：MVD=EndotoxinlimitconcentrationoftestsolutionMVD=內(nèi)毒素限值供試品溶液濃度/Endotoxinlimit:theendot

19、oxinlimitforactivesubstancesadministeredparenterally,definedonthebasisofdose,isequaltoKM內(nèi)毒素限值：在劑量的基礎(chǔ)上，注射藥物的活性成分的內(nèi)毒素限度為K/MK=thresholdpyrogenicdoseofendotoxinperkilogramofbodymass,M=maximumrecommendedbolusdoesofproductperkilogramofbodymass.K=人每公斤體重每小時(shí)最大可接受的內(nèi)毒素劑量（EU/kg）M人用每公斤體重每小時(shí)的最大供試品劑量。Whentheproduc

20、tistoeinjectedatfrequentintervalsorinfusedcontinuously,Misthemaximumtotaldoseadministeredinasinglehourperiod.Theendotoxinlimitforactivesubstancesadministeredparenterallyisspecifiedinunitssuchas:IU/mL,IU/mg,IU/Unitofbiologicalactivity,ect,inmonographs.專論中，注射劑活性成分的內(nèi)毒素限度的單位有IU/ml、IU/mg、IU/生物活性單位等。Conce

21、ntrationoftestsolution:供試品溶液的濃度表示法：mg/mLiftheendotoxinlimitisspecifiedbymass(IU/mg),當(dāng)內(nèi)毒素限度以質(zhì)量（IU/mg）表示時(shí)，用mg/ml表示濃度。Units/mLiftheendotoxinlimitisspecifiedbyunitofbiologicalactivity(IU/Unit),當(dāng)內(nèi)毒素限度以（IU/ml）表示時(shí)，用ml/ml表示濃度。ml/mLiftheendotoxinlimitisspecifiedbyvolume(IU/mL).當(dāng)內(nèi)毒素限度以生物單位（IU/Unit）表示時(shí)，用Units/

22、ml表示濃度。=thelablelledlysatesensitivityinthegel-clottechniques(IU/mL)orthelowestconcentrationusedinthestandardcurveoftheturbidimetricorchromogenictechniques.=指凝膠法中鱟試劑的標(biāo)示靈敏度，或指濁度法或顯色法使用的標(biāo)準(zhǔn)回歸曲線上最低點(diǎn)（IU/ml)的對(duì)應(yīng)值。7.GEL-CLOTTECHNIQUE(METHODSAANDB)凝膠法（方法A和B）Thegel-clottechniquesallowdetectionorquantificationo

23、fendotoxinsandisbasedonclottingofthelysateinthepresenceofendotoxins.Theminimumconcentrationofendotoxinsrequiredtocausethelysatetoclotunderstandardconditionsisthelabeledlysatesensitivity.Toensureboththeprecisionandvalidityofthetest,confirmthelabeledlysatesensitivityandperformthetestforinterferingfact

24、orsasdescribedunder1.Preparatorytesting.凝膠法系通過鱟試劑與內(nèi)毒素產(chǎn)生凝集反應(yīng)的原理來檢測(cè)或定量內(nèi)毒素的方法。在標(biāo)準(zhǔn)條件下，可引起鱟試液凝集的內(nèi)毒素的最低濃度即為鱟試劑的標(biāo)示靈敏度。為確保試驗(yàn)結(jié)果精確、有效，應(yīng)根據(jù)預(yù)備試驗(yàn)中的說明開展試驗(yàn)，復(fù)核鱟試劑的標(biāo)示靈敏度和試驗(yàn)的干擾因素。1.PREPARATORYTESTING預(yù)備試驗(yàn)(i)Confirmationofthelabeledlysatesensitivity鱟試劑的標(biāo)示靈敏度復(fù)核試驗(yàn)Confirmin4replicatesthelabeledsensitivity,expressedinIU/mL,

25、ofthelysatesolutionpriortouseinthetest.Confirmationofthelysatesensitivityiscarriedoutwhenanewlotoflysateisusedorwhenthereisanychangeinthetestconditionswhichmayaffecttheoutcomeofthetest.靈敏度用IU/ml表示。應(yīng)在鱟試劑用于檢查內(nèi)毒素之前對(duì)靈敏度進(jìn)行復(fù)核；復(fù)核試驗(yàn)需要制備標(biāo)示靈敏度的4支平行管。當(dāng)使用新批號(hào)的鱟試劑或試驗(yàn)條件發(fā)生了任何可能影響檢驗(yàn)結(jié)果的改變時(shí)，應(yīng)進(jìn)行鱟試劑靈敏度復(fù)核試驗(yàn)。Preparestanda

26、rdsolutionsofatleast4concentrationsequivalentto2,0.5and0.25bydilutingthestandardendotoxinstocksolutionwithwaterforBET.用BET用水稀釋內(nèi)毒素儲(chǔ)備標(biāo)準(zhǔn)溶液，制成2、0.5和0.25四種濃度的標(biāo)準(zhǔn)溶液。Mixavolumeofthelysatesolutionwithanequalvolumeof1ofthestandardsolutions(suchas0.1mLaliquots)ineachtube.Whensingletestvialsorampoulescontaining

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

28、vertitthroughapproximately180?inonesmoothmotion.Ifafirmgelhasformedthatremainsinplaceuponinversion,recordtheresultaspositive.Aresultisnegativeifanintactgelisnotformed.在每支試管中，分別混合等體積（通常為0.1mL)的鱟試液和標(biāo)準(zhǔn)溶液。如使用的是裝有鱟試劑凍干粉末的西林瓶或安瓿，可向其中直接加入溶液。按照鱟試劑生產(chǎn)商的建議，將反應(yīng)混合物孵育一段時(shí)間(通常在371下保溫602min)，在此過程中，不能振動(dòng)試管。凝膠的完整性測(cè)試：如使

29、用試管作為容器，先將試管從保溫箱中取出，再緩緩將試管倒轉(zhuǎn)180o。如果形成了堅(jiān)實(shí)的凝膠，倒轉(zhuǎn)后凝膠不移位，此時(shí)將該項(xiàng)檢查的結(jié)果記錄為陽性。如未能形成堅(jiān)實(shí)且不變形的凝膠，該項(xiàng)檢查的結(jié)果即為陰性。Thetestisconsideredvalidwhenthelowestconcentrationofthestandardsolutionsshowsanegativeresultinallreplicatetests.Theend-pointisthelowestconcentrationintheseriesofdecreasingconcentrationsofstandardendotoxin

30、thatclotsthelystae.Determinethegeometricmeanend-pointconcentrationbycalculatingthemeanofthelogarithmsoftheend-pointconcentrationsofthe4dilutionseries,taketheantilogarithmofthisvalue,asindicatedbythefollowingexpression:僅在最低濃度的標(biāo)準(zhǔn)溶液的所有平行管的檢查結(jié)果均為陰性的情況下，試驗(yàn)方為有效。反應(yīng)終點(diǎn)濃度指系列遞減的內(nèi)毒素濃度中最后一個(gè)呈陽性結(jié)果的濃度。將終點(diǎn)濃度取對(duì)數(shù)，計(jì)算它們

31、的平均值，再將平均值的結(jié)果取反對(duì)數(shù)，最后的表達(dá)式如下：Geometricmeanend-pointconcentration=antilogefe=sumofthelogend-pointconcentrationsofthedilutionseriesused,f=numberofreplicates.終點(diǎn)濃度的幾何平均值log-1(e/f)e所用系列溶液的終點(diǎn)濃度的對(duì)數(shù)值的和f平行管的數(shù)量Thegeometricmeanend-pointconcentrationisthemeasuredsensitivityofthelysatesolution(IU/mL).Ifthisisnotle

32、ssthan0.5andnotmorethan2,thelabeledsensitivityisconfirmedandisusedinthetestperformedwiththislysate.反應(yīng)終點(diǎn)的濃度的幾何平均值即為鱟試劑靈敏度的測(cè)量值（IU/ml）。當(dāng)終點(diǎn)濃度的幾何平均值在0.5至2.0之間時(shí)，可判定受試鱟試劑的標(biāo)示靈敏度為，可用于內(nèi)毒素的檢查。(ii)Testforinterferingfactors干擾因素試驗(yàn)PreparesolutionsA,B,CandDasshowninTable2.6.14-1,andusethetestsolutionsatadilutionles

33、sthantheMVD,notcontaininganydetectableendotoxins,operatingasdescribedunder1.preparatorytesting,(i)Confirmationofthelabeledlysatesensitivity.按表2.6.14.-1制備溶液A、B、C、D。供試品的稀釋度不得超過MVD，且供試品溶液不能檢查出內(nèi)毒素，具體操作見（1）預(yù)備試驗(yàn)，（i）鱟試劑標(biāo)示靈敏度的復(fù)核試驗(yàn)項(xiàng)。Thegeometricmeanend-pointconcentrationsofsolutionsBandCaredeterminedusingthe

34、expressiondescribedin1.Preparatorytesting,(i)Confirmationofthelabeledlysatesensitivity.根據(jù)（1）預(yù)備試驗(yàn)下的（i）鱟試劑標(biāo)示靈敏度的復(fù)核試驗(yàn)項(xiàng)中列出的公式，計(jì)算B和C溶液的終點(diǎn)濃度的幾何平均值。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溶液內(nèi)毒素濃度/配制內(nèi)毒素的溶液稀釋劑稀釋倍數(shù)稀釋后內(nèi)毒素的濃度平行管數(shù)A無/供試品溶液4B2/供試品溶液供試品溶液1248210.50.254444C2/BET用水BET用水1248210.50.252222D0/BET用水2A=經(jīng)檢查無內(nèi)毒素的溶液B=干擾實(shí)驗(yàn)用C=鱟試劑標(biāo)示靈敏度的對(duì)照品D=陰性對(duì)照品（BET檢查用水）Thetestforinterferingfactorsmustberepeatedwhenanychangesaremadetotheexperimentalconditionsthatarelikelytoinfluencetheresultofthet

37、est.如試驗(yàn)條件發(fā)生了任何可能影響到試驗(yàn)結(jié)果的變化，須重新進(jìn)行干擾因素試驗(yàn)。ThetestisconsideredvalidwhenallreplicatesofsolutionsAandDshownoreactionandtheresultofsolutionCconfirmsthelabeledlysatesensitivity.只有當(dāng)溶液A和D的所有平行管中無反應(yīng)、且溶液C的結(jié)果在復(fù)核的鱟試劑靈敏度范圍內(nèi)時(shí)，試驗(yàn)方有效。IfthesensitivityofthelysatedeterminedwithsolutionBisnotlessthan0.5andnotgreaterthan2

38、,thetestsolutiondoesnotcontaininterferingfactorsundertheexperimentalconditionsused.Otherwise,thetestsolutioninterfereswiththetest.經(jīng)測(cè)定，如果溶液B的靈敏度在0.5，2.0間，可判定供試品溶液在該實(shí)驗(yàn)條件下，對(duì)實(shí)驗(yàn)無干擾；反之則判定供試品溶液對(duì)試驗(yàn)?zāi)苄纬筛蓴_。IfthepreparationbeingexaminedinterfereswiththetestatadilutionlessthantheMVD,repeatthetestforinterferingfa

39、ctorsusingagreaterdilution,notexceedingtheMVD.Theuseofamoresensitivelysatepermitsagreaterdilutionofthepreparationbeingexaminedandthismaycontributetotheeliminationofinterference.若供試品溶液在小于MVD的稀釋倍數(shù)下對(duì)試驗(yàn)有干擾，應(yīng)將供試品溶液進(jìn)行不超過MVD的進(jìn)一步稀釋，再重復(fù)干擾試驗(yàn)。使用靈敏度更高的鱟試劑進(jìn)行內(nèi)毒素的檢查時(shí)，因?yàn)楦哽`敏度鱟試劑能排除實(shí)驗(yàn)的干擾因素，供試品的稀釋倍數(shù)也可適當(dāng)放寬。Interferenc

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

41、hhasthenbeensubmittedtothechosentreatment.可通過其他適宜的方法（如過濾、中和、透析或加熱處理等）排除干擾。為確保所選擇的處理方法能有效地排除干擾且不會(huì)使內(nèi)毒素失去活性，要使用預(yù)先添加了標(biāo)準(zhǔn)內(nèi)毒素再經(jīng)過處理的供試品溶液進(jìn)行干擾實(shí)驗(yàn)。2.LIMITTEST(METHODA)限度試驗(yàn)（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制備溶液A、B、C、D。實(shí)驗(yàn)方法見（1）預(yù)備試驗(yàn)的（i）鱟試劑標(biāo)示靈敏度的復(fù)核試驗(yàn)項(xiàng)。Table2.6.14.-2SolutionEndotoxinconcentration/SolutiontowhichendotoxinisaddedNumberofreplicatesANone/Dilutedtestsolution2B2/Dilutedtestsolution2C2/WaterforBET2DNone/WaterforBET2表2.6.14-2

43、溶液內(nèi)毒素濃度/配制內(nèi)毒素的溶液平行管數(shù)A0/供試品溶液2B2/供試品溶液2C2/BET用水2D0/BET用水2PreparesolutionAandsolutionB(positiveproductcontrol)usingadilutionnotgreaterthantheMVDandtreatmentsasdescribedin1.Preparatorytesting,(ii)Testforinterferingfactors.SolutionsBandC(positivecontrols)containthestandardendotoxinataconcentrationcorres

44、pondingtotwicethelabeledlysatesensitivity.SolutionD(negativecontrol)consistsofwaterforBET.制備溶液A、B（供試品陽性對(duì)照），稀釋倍數(shù)不得超過MVD。按照（1）預(yù)備實(shí)驗(yàn)，（ii）干擾因素實(shí)驗(yàn)項(xiàng)下的說明開展實(shí)驗(yàn)。溶液B、C（陽性對(duì)照）所含標(biāo)準(zhǔn)內(nèi)毒素的濃度為鱟試劑標(biāo)示靈敏度的兩倍。溶液D（陰性對(duì)照）為BET用水。(ii)Interpretation結(jié)果判斷ThetestisconsideredvalidwhenbothreplicatesofsolutionBandCarepositiveandthoseofs

45、olutionDarenegative.只有溶液B和C的平行管的試驗(yàn)結(jié)果均為陽性、溶液D的平行管的試驗(yàn)結(jié)果均為陰性時(shí)，試驗(yàn)方有效。WhenanegativeresultisfoundforbothreplicatesofsolutionA,thepreparationbeingexaminedcomplieswiththetest.溶液A的平行管的試驗(yàn)結(jié)果均為陰性時(shí)，可判定供試品符合試驗(yàn)的內(nèi)毒素限度要求。WhenapositiveresultisfoundforbothreplicatesofsolutionA,thepreparationbeingexamineddoesnotcomplyw

46、iththetest.溶液A的平行管的檢查結(jié)果均為陽性時(shí)，可判定供試品不符合試驗(yàn)的內(nèi)毒素限度要求。WhenapositieresultisfoundforonereplicateofsolutionAandanegativeresultisfoundfortheothers,repeatthetest.當(dāng)溶液A的平行管其中一個(gè)檢查結(jié)果為陽性，另一個(gè)為陰性時(shí)，重復(fù)試驗(yàn)。Intherepeattest,thepreparationbeingexaminedcomplieswiththetestifanegativeresultisfoundforbothreplicatesofsolutionA.

47、ThepreparationdoesnotcomplywiththetestifapositiveresultisfoundforoneorbothreplicatesofsolutionA.在重復(fù)試驗(yàn)中，溶液A的平行管的試驗(yàn)結(jié)果均為陰性時(shí)，可判定供試品符合試驗(yàn)的內(nèi)毒素限度要求。溶液A的平行管的檢查結(jié)果有一個(gè)或均為陽性時(shí)，可判定供試品不符合試驗(yàn)的內(nèi)毒素限度要求。However,ifthepreparationdoesnotcomplywiththetestatadilutionlessthantheMVD,thetestmayberepeatedusingagreaterdilution,no

48、texceedingtheMVD.但是，如果供試品不符合規(guī)定是在供試品的稀釋倍數(shù)小于MVD的情況下，應(yīng)將供試品溶液稀釋至較大但不超過MVD的倍數(shù)，再次開展試驗(yàn)。3.QUANTITATIVETEST(METHODB)(i)ProcedureThetestquantifiesbacterialendotoxinsinthetestsolutionbytitrationtoanend-point.PreparesolutionsA,B,CandDasshowninTable2.6.14.-3,andtestthesesolutionsaccordingtotheproceduredescribedu

49、nder1.Preparatorytesting,(i)Confirmationofthelabelledlysatesensitivity.通過將供試品滴定至反應(yīng)終點(diǎn)，對(duì)供試品所含的細(xì)菌內(nèi)毒素定量。按表2.6.14-3制備溶液A、B、C、D。具體實(shí)驗(yàn)操作見（1）預(yù)備試驗(yàn)的（i）鱟試劑標(biāo)示靈敏度的復(fù)核試驗(yàn)項(xiàng)。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溶液內(nèi)毒素濃度/配制內(nèi)毒素的溶液稀釋劑稀釋倍數(shù)稀釋后內(nèi)毒素的濃度平行管數(shù)A無/供試品溶液BET用水1248-2222B2/供試品溶液122C2/BET用水BET用水1248210.50.252222D無/BET用水2溶液A=不超過MVD并且通過干擾試驗(yàn)的供試品溶液。從通過干擾試驗(yàn)的稀釋倍數(shù)開始，用BET用水稀釋至1倍、2倍、4

54、倍和8倍，每一濃度準(zhǔn)備兩份稀釋液，最后的稀釋倍數(shù)不得超過MVD，并且確定稀釋液通過了干擾試驗(yàn)。也可以準(zhǔn)備其它合適倍數(shù)的稀釋液。溶液B=2濃度標(biāo)準(zhǔn)內(nèi)毒素的溶液A（供試品陽性對(duì)照）溶液C=含有2、0.5和0.25濃度標(biāo)準(zhǔn)內(nèi)毒素的BET用水系列，每一濃度準(zhǔn)備兩份。溶液D=BET用水（陰性對(duì)照）(ii)CalculationandinterpretationThetestisconsideredvalidwhenthefollowing3conditionsaremet:(a)bothreplicatesofsolutionD(negativecontrol)arenegative,(b)bothre

55、plicatesofsolutionB(positiveproductcontrol)arepositive,(c)thegeometricmeanend-pointconcentrationofsolutionCisintherangeof0.5to2.(ii)計(jì)算和結(jié)果判斷符合下列三個(gè)條件時(shí)，可將試驗(yàn)判為有效：溶液D（陰性對(duì)照）的兩組平行管均顯陰性，（b）溶液B（供試品陽性對(duì)照）的兩個(gè)平行管均顯陽性，（c）溶液C的終點(diǎn)濃度的幾何平均值在0.52之間。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.測(cè)定溶液A的內(nèi)毒素濃度，計(jì)算溶液A的系列稀釋液的終點(diǎn)濃度時(shí)，應(yīng)將內(nèi)毒素濃度記為終點(diǎn)稀釋倍數(shù)乘以。供試品的內(nèi)毒素濃度指這些平行管的反應(yīng)終點(diǎn)濃度的幾何平均值。如試驗(yàn)使用的是供試品的稀釋液，計(jì)算時(shí)，將檢查結(jié)果乘以稀釋倍數(shù)，即得到原

59、始溶液的內(nèi)毒素濃度。如在有效的試驗(yàn)中，供試品的檢查結(jié)果均顯陰性，應(yīng)記為內(nèi)毒素濃度小于（如實(shí)驗(yàn)使用的是供試品的稀釋液，檢查結(jié)果應(yīng)記為內(nèi)毒素濃度小于乘以供試品的最小稀釋倍數(shù)）。如供試品的所有結(jié)果均為陽性，應(yīng)記為內(nèi)毒素濃度大于或等于最大的稀釋倍數(shù)乘以（如，在表2.6.14-3中，初始稀釋倍數(shù)8）。如供試品內(nèi)毒素的濃度小于其專論中的規(guī)定濃度，可判定供試品符合檢查要求。ETRICQUANTITATIVETECHNIQUES(METHODSC,D,EANDF)8.光度測(cè)定法（方法C、D、E和F）（1）.TURBIDIMETRICTECHNIQUE(METHODSCANDF)（1）濁度法（方法C和F）Thi

60、stechniqueisaphotometrictesttomeasuretheincreaseinturbidity.Basedonthetestprincipleemployed,thistechniquemaybeclassifiedasbeingeithertheend-point-turbidimetrictestorthekinetic-turbidimetrictest.本法是利用光學(xué)試驗(yàn)來測(cè)定濁度增加的方法。根據(jù)檢測(cè)原理，這種方法又可分為終點(diǎn)濁度法和動(dòng)態(tài)濁度法。Theend-point-turbidimetrictest(MethodF)isbasedonthequantita