2.7.5. ASSAY OF HEPARIN - National Institute of …

1 Pharmeuropa : PA/PH/Exp. 6/T (12) 39 ANPNOTE ON THE GENERAL CHAPTERIt is proposed to replace the clotting ASSAY in sheep plasma by more specific chromogenic assaysfor anti-factor IIa activity and anti-factor Xa activity. To prepare for this revision, HEPARIN sodiumBRP has been recalibrated in a collaborative study, to be used with the new methods. In addition,the impact of this recalibration has been assessed by HEPARIN ASSAY OF , :a)directvisualinspection,preferablyusin gindirectilluminationandviewingagainstam attblackbackground;b)spectrophotometricr ecordingofthechangeinopticaldensityatawa velengthofapproximately600nm;c)visualdet ectionofthechangeinfluidityonmanualtilti ngofthetubes;d)mechanicalrecordingofthec hangeinfluidityonstirring, , ,andthatobtainedwiththehighestconcentrat ionissuchastogiveasatisfactorylogdose-re sponsecurve, ,labellingtheminduplicate:T1,T2andT3fort hedilutionsofthepreparationtobeexamineda ndS1, , ,S2,S3,T1,T2,T3,transfereachtubetoawater -bathat37 C,allowtoequilibrateat37 Cforabout15minandaddtoeachtube1mLofasuit ableAPTT(ActivatedPartialThromboplastinT ime)reagent(1)containingphospholipidanda contactactivator, ,using1mLofa9g/Lsolutionofsodiumchloride Rinplaceofoneoftheheparindilutions.

2 The2blankvaluesobtainedshould(1) Pharmeuropa , ,T2,T3,S1,S2, ( ). , ,combiningtheresultsoftheseassays,bytheu sualstatisticalmethods( ).Whenthevarianceduetodifferencesbetween assaysissignificantatP= , ,factorIIa(anti-IIaassay), ,calibratedinInternationalUnitsbycompari sonwiththeInternationalStandardusingthe2 assaysgivenbelow, ,carryouttheassaybydeterminingtheabsorba nce(end-pointmethod)orthechangeofabsorba nceperminute(kineticmethod)usingtubes, ,ofthesubstancetobeexaminedandofheparins odiumBRPintris(hydroxymethyl) ; :T1,T2,T3,T4foreachofthe4seriesofdilutio nsofthesubstancetobeexaminedandS1,S2,S3, LofantithrombinIIIsolutionR5and50 Lofeithertris(hydroxymethyl) , ,S2,S3,S4,T1,T2,T3,T4,T1,T2,T3,T4,S1,S2, S3,S4,allowtoequilibrateat37 C(water-bathorheatingblock)foratleast1mi nandaddtoeachtube25 ,transferthemixturestosemi-microcuvettes andmeasurethechangeinabsorbanceperminute ( ) ,stopthereactionafterexactly4minbyadding 50 Lofa20percent(V/V) ,ifnecessary, ,transferthemixturestosemi-microcuvettes andmeasuretheabsorbance( ) ,usingtris(hydroxymethyl) ; ,andcalculatethepotencyofthesubstancetob eexaminedinInternationalUnitspermillilit reusingtheusualstatisticalmethodsforpara llel-lineassays( ).

3 ,ofthesubstancetobeexaminedandofheparins odiumBRPintris(hydroxymethyl) ; Pharmeuropa :T1,T2,T3,T4foreachofthe4seriesofdilutio nsofthesubstancetobeexaminedandS1,S2,S3, LofantithrombinIIIsolutionR6and50 , ,S2,S3,S4,T1,T2,T3,T4,T1,T2,T3,T4,S1,S2, S3,S4,allowtoequilibrateat37 C(water-bathorheatingblock)for1minandadd toeachtube100 ,transferthemixturestosemi-microcuvettes andmeasurethechangeinabsorbanceperminute ( ) ,stopthereactionafterexactly4minbyadding 50 Lofa20percent(V/V) ,ifnecessary, ,transferthemixturestosemi-microcuvettes andmeasuretheabsorbance( ) ,usingtris(hydroxymethyl) ; ,andcalculatethepotencyofthesubstancetob eexaminedinInternationalUnitspermillilit reusingtheusualstatisticalmethodsforpara llel-lineassays( ).ReagentsTris(hydroxymethyl) , (hydroxymethyl)aminomethaneR, (hydroxymethyl) , (hydroxymethyl) (hydroxymethyl) , (hydroxymethyl)