In this issue: p€iJLAL UPDATE®

1 Inthisissue:MVDandMVCe-mailaddressCalend arVolume13, LAL User, this hasbeenayearofgreatsignificanceforAssoci atesofCape Cod,Inc. Dr. Watson'spassingwas a timeofsadness andintrospectionforus all. Animportantpartofthisyear'swork has beenadjusting to the lossofour ,partofDr. Watson's impres-sive legacy was asoundcompanybasedonqualityproductsandst affedbydedicatedpeople. It is now our task tobuildon in inEurope thisyearhas been newsubsidiarycompanies,ACCIE urope andACCIUK have done outstand-ing work. Here in the US, weaddedto ourtechnicalstaffandran more trainingcourses in1995than ever before. Becauseofthedemandfortraining courses, we areadding a new training center onto is a new bottling suiteformulti-testvials which will be complete by softwareforthe LAL-5000 has been very wellreceivedsince itsreleaseearlierthis year.

2 We have anumberofresearch anddevelopmentirons in thefire and will keep youinformedwhen newproductsare arelookingforwardto the challengesof1996and to an exciting atAssociatesofCapeCodwishyou andyourfamiliesHappyHolidays andthe very ,/~/;7~ , iJLALUPDATE , LAL test, regardlessofmethod, is usually more sensitive than isnecessary to detect the endotoxin limitofa thereforebe diluted to overcome interference while still allowing detectionoftheendotoxin limit. Dilution is by far the mostimportantstrategyfor dealingwith interference. However, aproductmay not be diluted beyond the pointofbeing able to detect the endotoxin limit. this dilution is called themaximum valid dilution or MVD. The MVD is a dilution represents a Ipartin 20partstotal dilution. The minimum validconcentration(MVC) is theproductconcentrationat the know the MVDand/orMVC when confronted with a newproductto test or significanceofthe MVD is bestillustratedby an limitofproductA is 5 EU/ml and the test sensitivity is EU/ml, the method is clearly much more sensitive than isnecessaryto detect twenty times more sensitive than necessary.

3 Thisfactoroftwentyis the contaminatedwith an endotoxinconcentrationof5 EU/ml, it should fail the test. When thisproductis diluted by a factoroftwenty, the endotoxinconcentrationin the dilution is EU/ml, whichis detectable and theproductwill fail the test. Atgreaterdilutions, theendotoxin will not be detectable. A negative test at a dilutiongreaterthan theMVD gives noassurancethattheproductcontains less than the , a positive gel-clot testofundilutedproductA using a EU/ml does notrepresenta failure. Such a test result onlymeans that theproductcontains more than EU/m!. It may still containless than 5 EU/m!. In order to determine whether theproductcontains equalto or more than the limitconcentration,it must be diluted to the this reasonthatthe test forBacterialEndotoxins in the EuropeanPharmacopeia(EP) states that samples should be tested at the MVD.

4 Onlywhen testing at the MVD are you performing apass/failtest by the gel-clotmethod. (The EP and otherpharmacopeiado not detail procedures for theturbidimetricor chromogenic methods). 1995,Associatesof Cape Cod, Inc.,WoodsHole,MassachusettsAll rightsreservedFormulaeforCalculationofMV DE xampleofMVDC alculationThe simplest formula for MVD appliesifthe endotoxinlimit isexpressedin EU/ml. this is the formula given in theEP, which refers to the endotoxin limitconcentration(ELC)expressed in EU/ml. (See LAL update , June 1995 fordiscussionofendotoxin limits).To continue with the example given above in whichproductA has a limitof5 EU/ml and the LAL has asensitivity(A) :MVD=5 EU/ml= EU/mlMVD=Endotoxin limit in EU/mlAAssume limit forproductA is EU/mg and theconcentrationis 100 mg/ml, thenwhereA(lambda) is the sensitivityofthe test (see box fordetails).

5 MVD= EU/mg x 100 mg/ml= EU/mlIfthe endotoxin limit isexpressedin units other than EU/ml( EU/mg or EU/IU), theproductconcentration(orpotency) must be included in the is theequation given in the FDA Guideline as Method I forcalculatingthe isimportantto notethatMVD increases as the sensitivityofthe test reagent sensitivityofthe LALreagent used is EU/ml for the aboveproduct(a reagentthatis four times more sensitive) the MVD increases by limit in EU/ml x product concentrationAMVD=5 EU/ml= EU/mlNote the EU/ml in thecalculation,eventhough the sensitivityofthe LAL on the vial label is given EU/mt. The additional decimal places are meaninglessin termsofLALsensitivity because there is an allowance fora twofold error in the method. However, they do tend to yieldround numbers.

6 Indeed, if is used for the divisor in theabovecalculations,we get insteadofa also a little more conservative to use theadditional decimal places. this matter wasdiscussedin theLALUPDATE, the MVD is thegreatestdilutionofproductatwhich the endotoxin limit can be detected, the MVC is thelowestconcentrationofproductat which the endotoxin limitis detectable in a given test method. Because it is aconcentration,the MVC is directlyapplicableto productswith endotoxin limitsexpressedin units such as EU/mg, EU/IV, EU/mEq, value forproductswithendotoxin limitsexpressedin EU/ml, in which case the MVCis expressedin the MVD is a unitless dilution factor, the MVChas unitsofconcentration. The mostimportantdistinctionbetween the two is that, for a given test sensitivity, the MVCisabsolutewhereas the MVD is influenced byproductconcentration.

7 Thus, two different potenciesofa productwill have different MVDs, but the MVC is bear this in mind when making up test solutionsfrom bulk powder; the MVD will depend upon the initialin ChromogenicandTurbidimeA Moveable FeastIn the equati(lamis usetest. In the cassimple;A isthe Iysthe lot of LAL reagn the anot fixed. If a sample is found table endotoxin in any, the rless than )theentration ofdard. Thus, the sensiti .y giis thest concentran used tostandard curve for that assay. Becauseof the standard curve can be changed, calculating MVD and MVC values formethods other than gel-clot, use for the value ofAthe lowest concentration that you could employ in astandard curve. this will givemaxipos-sible value for the MVD (andlowest p sibleMVC) for the method. Whether these extrefMVD or MVC, or indeed ofA,are actually uslikely to depend on the degree to which the printerferes with the test.

8 The degree of interferis determined , Number4, is this independence from the numberofthepharmacopeiachapterson endotoxintests refer to the MVD but none mention the MVC. Only theFDA Guideline refers to the MVCis the first step given to determine the MVD by method following equation assumesthatthe endotoxin limitofthe product has been determined, whereas the equation in theGuideline does limitExampleofMVCC alculationAgain, we will use ourhypotheticalproductA with anendotoxin EU/mg and an LAL EU/ml:MVC= EU/ml=5 EU/mgThe MVC is inversely related to the MVD. Thegreaterthedilutionofthe product, the lower the concentration. Theformula for converting an MVC to the MVD is:MVD=productconcentrationMVCE xample: Using the figures from our example:MVD=100 mg/ml=205 mg/mlBecause the MVC is inversely related to the MVD, anincreased sensitivityofthetest method reduces the MVC andincreases the , the more sensitive test allowsthe endotoxin limit to be detected at a lowerconcentration!

9 Greaterdilutionofthe conclusion, while the MVC is more rugged than theMVD because it is independentofthe initialproductconcentration, the conceptofMVD is more can becalculatedfor any samplethathas an endotoxin limit assigned to it. ForcalculationofMVDs, it is recommendedthatendotoxin limits beexpressedin or converted to EU/ml (or IU/ml). (See the article onendotoxin limits in the LALUPDATE,June 1995). this isthe EPapproachand it is a reasonable one because these arethe unitsoftestsensitivity the limit isin EU/ml, the conceptofMVDbecomes obvious. UseoftheLALUPDATEVol. 13, Number 4, page 3 MVD requires anunderstandingofthe concept, because anMVD is specific to , otherwise invalid test resultsare now have a web site where you canbrowsethroughourproductcatalog, the latest LAL UP-DATEand listingsofback issuses.

10 Send us LALS ingleTestVialsStarting in 1996, we will nolongerbeputting Mylar?" labels onPyrotell"LALsingletestvials (STVs).The vial will havethe lot number printed directly on the glassby an inkjetdot printerofthe type used toprint expiration dates on many food contain-ers. Thepackagewill still be labelled withthe lot specific information: lot number,sensitivity, and expiration date. this changewill increase our efficiency and give usbettercontrolofthepackagingprocessand costs.