GMP如何建立清潔驗證可見殘留限度

如何建立清潔驗證可見殘留限度Abstract摘要Visibleresiduelimits(VRLs)havebeenshowntobeavaluabletoolinavalidatedcleaningvalidationprogram.可見殘留限度(VRLs)已經(jīng)成為了清潔驗證過程中一個有價值的工具。Visibleresiduelimits(VRLs)canbequantitativelyestablishedforpharmaceuticalproducts,APIs,excipients,anddetergents(1-3).TheVRListhelowestlevelofresiduethatcanbeseenbyapanelofobserversunderadefinedsetofobservationparameters.IfaVRLislowerthanthecalculatedacceptablecleaningresiduelimitforapieceofequipmentoranequipmenttrain,thentheVRLcouldbeconsideredthecleaninglimitforthatequipment.TheregulatoryagencieshaveacceptedtheuseofVRLsasavaluabletooltohelpdevelop,validate,andmaintainarobustcleaningvalidationprogram(4,5).對于藥品、API、輔料和清洗劑，可見殘留限度（VRLs）能夠被量化制定。VRL是殘留物的最低水平，這些殘留物在一套設(shè)定好的檢測參數(shù)下能夠通過檢測儀表設(shè)備能被看到。如果對于一個設(shè)備或設(shè)備組件， VRL低于計算出的接受清潔殘留限度，那么VRL應(yīng)該被認為是設(shè)備的清洗限度。監(jiān)管機構(gòu)已接受使用VRLs作為一個有價值的工具去開發(fā)、驗證和維護一個健全的清洗驗證程序。TheruggednessofVRLshasbeenestablishedfordifferentresidues,personnelandviewingconditions(6),presentationofcouponsforpersonnelobservation(7),andfordifferentmaterialsofconstruction(MOCs)(8).Throughallofthesestudies,themethodofpreparationoftheVRLsampleswasheldconstant.Asolutionorsuspensionwaspreparedinavolatilesolvent,typicallymethanol,serialdilutionsweremadetodecreasingconcentrationlevelsandcouponswerespikedbypipetting100pLontotheMOCcoupon.Themethanolspreadouttoacircleofabout5-cmdiameterandrapidlyevaporated,leavingaringofvisibleresidue.對不同殘留物、人員、觀察條件和觀察樣本、不同材質(zhì)條件下的 VRLs進行了研究。通過這些研究，VRL樣品制備方法一直保持不變。溶液或懸浮液在揮發(fā)性溶劑中制備，代表性的是甲醇，經(jīng)過一系列的稀釋濃度水平逐漸降低，并且取樣片被加入標定物，通過加入 100同不同化工材料。這種甲醇在直徑5cm的圓形物上展開并且迅速揮發(fā)、留下一個可見殘留物的圓環(huán)。Measuringfullarea.圖1：測量全部區(qū)域Background背景表1:標的制備和目標濃度標定溶液制備（稀釋舉例）標定溶液濃度被標定體積被標定的殘留物目標濃度取樣片（20cm2）Spikingpreparationandtargetconcentrations.表1：標的制備和目標濃度TheinitialworkonVRLs(1)startedwiththe100-pglevelwithatargetof4pg/cm2tocorrelatewiththelevelcitedintheFourmanandMullinarticle(10).Additionallevelsatdecreasingvalueswerealsodetermined,butatruevisuallimitwasnotdeterminedifthelowestleveltestedwasvisibleanditwassufficientlylowerthantheacceptablecleaningresiduelimit(ARL)(i.e.,atleasttwotimesloweror50%oftheARL).TheinitialworksurveyedawiderangeofAPIs,excipients,andformulationsoverarelativelyshortperiodoftimetoestablishacompletedatabaseforacleaningvalidationprograminaclinicalmanufacturingfacility.AsapplicationsofVRLsexpanded(2)andexperiencegrew,thespikinglevelsoftheresidueswerelowereduntilafinalsetofspikelevelswerefinalized,asshowninTableI.VRLs最初的工作始于100-陽水平對應(yīng)4pg/cm2的目標。其他低于這個值得水平也被進行了測定，但是真實的可見限度有沒被測定，如果測試的最低水平是可視的并且足夠低于可接受的清潔殘留限度(例如，至少低兩倍或者可接受清洗限度(ARL)的50%)。最初工作調(diào)查APIs、輔料和配方的寬泛范圍，覆蓋一個相對較短的周期去建立一個完整數(shù)據(jù)庫為臨床生產(chǎn)設(shè)施的清洗驗證程序。VRLs應(yīng)用程序擴充和經(jīng)驗的增長，殘留物的標定水平在不斷降低直到最后設(shè)定標定物水平定案，如表1所示。TheaverageofseveralhundredVRLdeterminationswas1.1|ig/cm2(6),whichmadeitapracticalandachievablecorrelationtousetheVRLastheARL,althoughitwasnotedthatroughlytwo-thirdsoftheVRLswereabletobeseenatthelowestlevelspikedontothecoupons.Therewasaclearindicationofthecurrentsituation.Inaddition,theaveragenumberswerealittlemisleadinginthatasexperienceincreasedindeterminingtheVRLlevels,thenumbersgeneratedbegantoskewtolowerlevels(6).Now,when1pgofmaterialisspikedontoastainlesssteelcouponandallowedtospreadouttoa5-cmdiametercircle,itisoftenstillvisibleunderdefinedconditionsofviewingdistance,viewingangle,andlightlevel.Theareaofthesamplewitha5-cmdiameteris19.6cm2(nx(2.5cm)2),makingtheconcentrationofthesample1pg/20cm2or0.05pg/cm2.Thiswouldequatetoaswabsampleof1.2pgusinga25-cm2swabarea.Extractingthe1.2-pgsampleintosolventforanalysisresultsinasolutionconcentrationwellbelow1.0pg/mL.幾百個VRL測試的平均值是1.1pg/cm2,VRL作為ARL使用是切合實際和可實現(xiàn)的相關(guān)，雖然值得注意的是，大致三分之二的 VRLs能夠被視為在取樣片上被最低水平標定。這明確的指出了當前的形勢。另外，平均值有一點誤區(qū)，因為在經(jīng)驗較少的情況下測定的 VRL水平可能使最低水平歪斜?，F(xiàn)在，當 1pg物質(zhì)被標定到無銹鋼取樣片并且允許在5cm直徑的圓形上展開，它在特定的距離、角度和燈光水平下觀察經(jīng)常仍然是可見。5cm直徑的樣品面積是19.6cm2(nx(2.5cm)2)，樣品濃度是1pg/20cm2或0.05pg/cm2。這等同于1.2pg樣品在25-cm2的面積上抽濾。提取1.2pg樣品到溶劑中去分析結(jié)果，溶液濃度遠低于1.0pg/mL。表II:標的制備和濃度標定溶液濃度被標定體積被標定的殘留物的直殘留物的面積（cm2）殘留物的濃度(pg/mL)(pL)殘留物（pg）徑（cm）(pg/cm2)Spikingpreparationandconcentrations.表2:標的制備和濃度Usinga1|ig/20cm2or0.05pg/cm2VRLasacleaninglimitisnotpractical.VRLsestablishedusingthisapproachatthislevelcouldresultinapracticalquantitativecontradictionbetweentheVRLandthelimitofquantitation/limitofdetection(LOQ/LOD)oftheanalyticaltestmethodiftheVRLislowerthantheLOQ/LOD.Perregulatoryguidance,thesensitivityoftheanalyticalmethodshouldextendbelowthecleaninglimittoassesstheeffectivenessofthecleaningprocedure.Thisapproach,however,isnotpossibleifthevisuallimitislowerthantheanalyticallimit.用1pg/20cm2或0.05pg/cm2的VRL作為清洗限度是不切合實際。用這個方法建立的VRLs將導致VRL與定量限/檢測限之間的矛盾。按照法規(guī)指南，分析方法的靈敏度必須低于清潔限度，以評估清洗程序的有效性。但是如果可視限度低于分析限度，那么這個方法是不可能的。Measuring'donut'area圖2:測試環(huán)形面積AslongastheVRLisabovetheLOQ/LODoftheanalyticalmethod,thisapproachtodeterminingVRLsisacceptable.However,aspersonnelgainexperience,theVRLsstarttrendingtolowervaluesandtheanalyticalsensitivitywillbecomeanissue.Eitheranalternative,morepracticalquantitativeapproachisnecessary,orVRLsgeneratedusingthisapproachcanbetreatedasmorequalitativewiththeassurancethattheequipmentwillbevisuallysoiledlongbeforethereisaconcernforpatientsafety.只要VRL在分析方法的LOQ/LOD以上，這個方法測試VRLs是可以接受的。但是隨著人員經(jīng)驗的增長，VRLs起始趨勢是較低的值并且分析敏感度將變成問題。任何一個選擇，較多的實際定量方法是必須的，或者用這個方法生成VRLs能夠被當作設(shè)備被可見污染的較多的定性和保證，在很久之前病人安全問題就對此進行過關(guān)注。Methodology方法學ThereareseveralalternativestotheVRLmethodologydescribedinthefirstpartofthisarticle,bothqualitativeandquantitative.Asecond,qualitativealternativewouldbetospikeresidueattheARLandatsomelevelbelowtheARL(50%or25%)usingthesamespikingprocedureasdescribedpreviously.Iftheresiduesarevisuallydetected,thentherewouldbeconfidencethattheresiduewouldbevisibleonequipmentatlevelslowerthantheARLand,therefore,visualinspectionwouldbeappropriatetodetermineequipmentcleanliness.TheVRLwouldbelistedas/lessthan’thelowestresidueleveltested.Forthisstudy,twoadditionalalternativemethodsofdeterminingpractical,quantitativeVRLswereexplored.AthirdapproachistocontinuetopreparetheresiduesamplesinthesamemannerasdescribedandmeasuretheareaofonlytheresidueringbysubtractingtheareaoftheinnercirclefromthelargercircleleavingonlytheareacoveredbytheresidueasshowninFigures2and3usingEquation1:[Eq.1]RingArea=nr2-nr22=n(r12-r22)=n(r1-r2)(rl-⑵在這篇文章的第一部分對VRL描述的方法學有幾個可選方案，定性和定量相結(jié)合。第二個定性選擇是標定殘留物在 ARL和ARL低一些的水平(50%或25%)，使用的標定過程如前所述。如果殘留物能夠被可視檢測，那么就有信心能夠檢測到設(shè)備上的低于ARL水平的殘留物，因此，目視檢查也適用于檢測清潔線的設(shè)備。 VRL被列為〃低于〃檢測到的最低殘留物水平。對于這項研究，測定實際定量的 VRLs的另外兩個可選方法進行了探索。第三個方法是按照描述持續(xù)制備同樣的殘留物樣品，通過大圓減去內(nèi)部小圓的面積僅留下被覆蓋的面積進行測定殘留物圓環(huán)的面積，殘留物如圖2和圖3所示用公式1:圓環(huán)面積=nr2-nr22=n(r12-r22)=n(r1-r2)(r1-r2)Measuring'donut'area.圖三：測定圓環(huán)面積Forexample,spiking100pLofa10-pg/mLspikingsolutionresultsinaresiduewithanouterdiameterof5cmandaninnerdiameterof4.8cm,theareaoftheresidueis1.5cm2comparedtotheareaofthe5-cmcirclealone,whichis19.6cm2.Fora1-pgresiduesample,thischangestheconcentrationfrom0.05pg/cm2to0.67pg/cm2.Thiswouldresultinaswabsampleof17pgusinga25-cm2swabarea.例如：取100同10pg/mL的標定溶液結(jié)果是一個外直徑5cm和內(nèi)直徑4.8cm的圓環(huán)的殘留物，圓環(huán)殘留物的面積僅是1.5cm2，相當于直徑5cm的圓，它的面積是19.6cm2。對于1陽的殘留物樣品，濃度從0.05pg/cm2變化到了0.67.g/cm2。這將導致17pg的抽濾樣品用于25cm2的抽濾面積。Afourthapproach,whichshouldalsobemorequantitative,istogeneratearesiduewithauniformcoverage.Becauselowspikelevelssuchas1pgin100pLresultinaringresidueatlargerareas,minimizingtheareaoftheresiduecanbeaccomplishedbyspikingaminimumvolumeofahigherconcentrationsample.Spiking20pLofa75pg/mLspikingsolutionresultedinamoreuniform1.5-pgresidue.Theresultinguniformresiduespreadoveracirclewithadiameterof0.8cmandanareaof0.5cm2andaconcentrationof3pg/cm2or75pg/25cm2.第四個方法也是比較偏重定量，是去用統(tǒng)一的覆蓋面形成殘留物。因為像1pg/100pL的低標水平導致圓環(huán)殘留物在較大的面積，較小殘留物的面積能夠通過高濃度樣品小體積的標定來實現(xiàn)。取 20pL75pg/mL的標定溶液導致1.5pg殘留物分布比較均一。由此產(chǎn)生的均勻殘留物遍布在一個直徑0.8cm的圓上，0.5cm2的面積和3pg/cm2或75pg/25cm2濃度。Measuringuniformarea.圖四：測試均勻面積Serialdilutionsweremadeandsimilarlyspiked.TheadditionalsamplesareshowninTableIIandFigure4.ItcanbeseeninFigure4thatthefirstthreespotsareuniformwhilespotsfourandfivearenotuniformlydistributed.TheVRLcanbedeterminedusingthelowestuniformlydistributedresiduefromspotthreeforaVRLof0.5pg/cm2.Althoughspotsfourandfivearevisible,theyarenotuniformlydistributedandattemptingtodetermineaVRLwouldresultinthesameissueaswiththeFigureldetermination(i.e.,overestimatingthearearesultinginalowerVRL).Thissituationoftheuniformandnon-uniformresiduesisanalogoustotheLOQandLODofananalyticaltestmethod.Thethirdspotcanbequantitativelydetermined,butquantitationofspotsfourandfiveisnotpossibleeventhoughtheyarevisible.系列稀釋制成類似的標。額外的樣品在表II和圖四中顯示。在圖四中能夠看到前三個斑點分布比較均勻，而第四、五個分布不均勻。 VRL能夠用最低均勻分布殘留進行計算，按照斑點三計算VRL是0.5pg/cm2o雖然斑點三和斑點四是可見的，但是它們分布分布不均勻，如果試圖計算VRL將導致圖一測定相同的問題(例如，評估過高的面積導致VRL較低)°均勻和不均勻殘留物的情況與LOQ和LOD分析測試方法相似。第三個斑點能夠被定量分析，但是斑點四和五的定量是不可能盡管他們是可見的。Discussion討論TableIII:Optionsforestablishingvisibleresiduelimits(VRLs).MeOHismethanol,ARLisacceptablecleaningresiduelimit,LOQislimitofquantitation.表三：建立可見殘留限度的選擇MeOH是甲醇，ARL是可接受的清潔殘留限度，LOQ是定量限。表三：建立可見殘留限度的選擇，MeOH是甲醇，ARL是可接受的清潔殘留限度，LOQ是定量限。VRL測定方法學方法1234“按現(xiàn)狀”-標定殘留物用100HL甲醇，測定所有面積“定量”-標定殘留物用100HL甲醇至1」50%ARL水平“修改現(xiàn)狀”-標定殘留物用100HL甲醇測量圓環(huán)面積“均勻覆蓋”-標定殘留物用較少的體積較高的濃度去確保均勻覆蓋制備制備簡單制備簡單制備簡單稍微較長的制備直到建立參數(shù)測定單次測量無二次測量單次測量精確度相對定量定性定量定量問題可能低于分析的不接近現(xiàn)實的V環(huán)的寬度主觀在低殘留物水LOQRL平，均勻覆蓋困難EachofthefourapproachesforVRLshasitsadvantagesaswellasdrawbacks.TableIIIprovidesasummaryforthedifferentapproachesincludingpreparation,levelofVRL,andprimaryissue.Eachisareasonableapproachaslongasthecompanydefinestheirprocessandisawareoftheintentandlimitationsfortheapproachtheyhavechosen.四個VRLs的方法每個都有自己的優(yōu)勢和缺點。表III對不同方法包括制備、VRL水平和主要問題進行了總結(jié)。每個方法都是合理的，只要公司規(guī)定自己的工藝過程并且清楚自己所選方法的意圖和局限性。Theinitialapproachdescribedinthebackgroundsectionofspikingaknownquantityofresidueindecreasingamountsontomultiplecouponsandthenmeasuringtheareaoftheentirecirculardriedresidueprovidesassurancethatvisibleresiduewouldbedetectedontheequipmentsurfaceswellbeforetheresiduebecomesasafetyriskinthesubsequentmanufacturedbatch.ThisdeterminationofaVRLprovidesaquantitativeassessmentofthe“marginofsafety”betweentheVRLlevelandtheARLofthespikedAPIorformulation.ThisapproachservesasamuchbettertrainingtoolinthatpersonnelwillgetabetterimpressionoftheappearanceoftheresiduesastheyneartheVRL,whichisavaluableperspectivetohave,particularlyforoldercleanedequipmentthatissubjecttootherpermanentmarksonthesurfaces.Itisalsopracticalinthatresiduewilloftenappearasringsorwaterspotsoncleaned,driedequipment.最初描述的方法在標定已知殘留物數(shù)量的背景章節(jié)中，在多個取樣片上減少數(shù)量，然后測量整個圓面積的干殘留物，保證可見殘留物能夠在設(shè)備表面可以很好的檢測，之前的殘留物對隨后的生產(chǎn)批次是一個安全的風險。VRL的測定在VRL水平和標定API或配方的ARL之間提供了〃安全邊緣〃定量評估。這個方法作為人員很好的培訓工具在VRL附近將獲得殘留物外觀更好的印象，這是一個有價值的視角，特別對有其他永久標記的較老的清洗設(shè)備表面。在殘留物經(jīng)常出現(xiàn)的圓環(huán)或清洗、干燥設(shè)備的水印也有實用性。ThemajorpotentialdrawbacktothisapproachcanbeseeninTableI.Ifa1-pgspikedsampleisvisible,whichisoftenthecase,itwillhavearesultingconcentrationof0.05pg/cm2.Swabbingthissampleandextractinginto10mLofsolventwouldresultinasampleconcentrationof0.1pg/mL.IfnotalreadybelowtheLOQoftheanalyticalmethod,itwouldbeveryneartoitformostanalyticaltestingtechnologies,includingthecommonlyusedtechnologiesfortestingcleaningsamples,suchashighperformanceliquidchromatography(HPLC)andtotalorganiccarbon(TOC)analysis.BecausetheVRLshouldbeconsideredequivalenttotheARL,itisexpectedthatthetestmethodforcleaningsamplesshouldhaveanLOQlowerthantheARLfortheresidue.Theanalyticaltestmethodmightnotbeadequatelysensitive,orifitissensitiveenough,sampleswouldbeindangeroffailingifanythingisdetectedinthetest.這個方法主要潛在的缺點在表1中能夠看到。如果1pg標定樣品是可視的，通常情況下，它將有一個0.05^g/cm2的最終濃度。擦拭樣品和萃取到10mL的溶劑中將出現(xiàn)0.1pg/mL的樣品濃度。如果這個值在分析方法的LOQ以下，大多數(shù)分析測定技術(shù)比較接近，包括通用的清潔樣品測定技術(shù)，例如高效液相色譜( HPLC)和總有機碳分析(TOC)。因為VRL被認為等同于ARL,所以希望清洗樣品的測試方法有一個比殘留物ARL更低的LOQ。分析測定方法可能不足夠敏感，或者敏感性足夠，所有東西在測定中都能檢測到，那么樣品面臨失敗的危險。ThisdangerofapotentialcleaningfailureusingthisVRLapproachisunjustifiedbecausethemajorityofthespiked,driedresidueareaisnotoccupiedwithresidue.Attheselowspikedconcentrations,aringisformedandtheresidueoccupiesonlyasmallareaaroundtheperimeterofthedriedcircle.AlthoughthisapproachtodeterminationofVRLsisefficientandprovidesanaccuratedeterminationoftheamountofresiduedividedbytheareaoftheresidue,theresidueisnotauniformresidueandresultsinaVRLlowerthanispractical.AVRLdeterminedthiswaycanbemisleadingifanattemptismadetorelateittotheLOQoftheanalyticalmethod.VRLsestablishedusingthisapproach,however,cancertainlybecomparedtotheADEcalculatedcleaninglimittodeterminethatvisualinspectionisadequateforcleaningconfirmation;andtodeterminehowwidethe“marginofsafety”isbetweenthevisuallimitandthecleaninglimit.潛在清洗失敗的危險使用這個VRL方法是不能被論證的，因為大多數(shù)標定物、干燥的殘留物面積沒有被殘留物占據(jù)。在低的標定濃度，形成圓環(huán)，殘留物僅占據(jù)干燥圓周長附近的小部分面積。雖然這個方法測定VRLs是有效的，并且提供了通過殘留物數(shù)量除以殘留面積得到準確的測定，但是殘留不均一導致VRL低于真實水平。VRL測定把這個方法帶入歧途，但是如果企圖把它和分析方法的 LOQ相關(guān)聯(lián)。不管怎樣，用這個方法建立的VRLs與ADE計算出的清潔限度比較確認可視性檢查適用于清洗確認；同時測定可視限度和清洗限度之間的〃安全邊界〃有多寬。Thesimplestapproachisthesecondonedescribedpreviously,whichistospikecouponsatasinglelevelthatislower(e.g.,50%)thantheARL.Ifthespikedresidueisvisible,itprovidesassurancethatvisibleresiduewouldbedetectedontheequipmentsurfacesbeforetheresiduebecameasafetyriskinthesubsequentmanufacturedbatch.Thisapproachavoidsthelowlevelpracticalityissueandalsohastheadvantageofspeedinthatsamplescanbepreparedrelativelyquicklyandcalculationsarenotnecessary.Fewercouponswouldbeusedperresidue,therefore,moreresiduescouldbequalifiedatthesametime.ThisapproachmightberecommendedifacompanywishedtoestablishaVRLprograminanefficienttimeframeorquicklydeterminethelimitationsofdetectingthevisibleresidueusingmultipledistancesandviewingangles.Thisapproachwouldalsobeusefulfortrainingorqualifyingalargegroupofpersonnel,suchasallequipmentwashersandinspectionpersonnel.Thisapproachisalsopracticalinthatresiduewillmostoftenappearasringsorwaterspotsoncleaned,driedequipment.之前描述的第二種方法最簡單，是直接標定取樣片在ARL更低的水平（例如50%）。如果標定的殘留物是可視的，為可視殘留物在設(shè)備表面被檢測提供保證，以前的殘留物成為了隨后生產(chǎn)批次的一個安全風險。這個方法避免了低水平的實際問題并且也有速度的優(yōu)勢，樣品制備比較迅速并且不需要計算。每個殘留使用的取樣片較少，因此，多個殘留物能夠同時定性。如果公司希望建立高效時間結(jié)構(gòu)或迅速測定的VRL程序用多個距離和視角檢測可視殘留物的限制，這個方法可以推薦。這個方法也用于培訓或很多人的資格確認，比如所有設(shè)備清洗人員和檢測人員。這種方法也實用于殘留物最常表現(xiàn)為環(huán)狀或清潔干燥設(shè)備上的水印。Thedrawbackswiththisapproacharethatitprovidesaqualitativedeterminationonly,whilenotwelldefiningthe“marginofsafety”betweentheVRLandtheARL.Aminordrawbackisifthespikedresidueisnotvisible,onecannotbecertainthatsomelevelbetweenthespikedlevelandtheARLmightstillbevisibleandadditionalcouponswouldhavetobespikeduntilavisualresidueisobserved.ThebiggestdrawbacktoapurelyqualitativeapproachisthatthevisuallimitshouldactuallybethecleaningARLifresidueisvisibleatlevelslowerthanthecalculatedcleaningARL.However,ifacompanystatesintheirstrategythattheyarelookingatVRLs,solelyfromaqualitativeperspectiveandthatthelowerspikelevelprovidesalevelofconfidencetoensurethatvisiblysoiledequipmentwillnotbedeemed‘clean,’andusesthecalculatedARLsastheircleaninglimit,thisshouldbeaviableapproachanddefensiblewiththeagencies.這個方法的缺點是它僅提供了定性測試，而不能確定 VRL和ARL之間的〃安全邊緣〃。次要的缺點是如果被標定殘留物不是可視的，不能確定ARL和被標定的水平之間的水平可能仍然是可見的，額外的取樣片不得不標定直到觀察到可視殘留物。對于純定性的方法最大的缺點是如果殘留物是可視的且水平低于計算出的清洗 ARL那么可視限度應(yīng)該是實際上的清洗ARL。但是，如果一個公司聲明他們在戰(zhàn)略上看看VRLs，僅從定性角度和較低的標定水平提供置信水平去確認被可視殘留物污染的設(shè)備不能認為是清潔的，用計算出的 ARLs作為他們的清潔限度，這應(yīng)該是可行的方法和站得住腳的中介。Thethirdapproachistousethesamespikingtechniqueasthefirsttwoapproaches,butthenmeasureonlytheringareathatiscoveredbytheresidue.Thereareseveraladvantageswiththisstrategy.Thespikingofthecouponsisstraightforward,theresiduesappearastheywouldoncleanedanddriedequipment,andmostimportantly,t