Characteriza*onofAc*veIngredients,By-Products,Impuri*es,andStandards

JeanneLink,PhD-OregonHealth&ScienceUniv.Disclosures:None“Everypharmaceu*calproducthasestablishediden*ty,strength,purity,andotherqualitycharacteris*csdesignedtoensuretherequiredlevelsofsafetyandeffec*veness”Ref:Cox

“Almosteverymachinewillgiveyouanumber”(Link)

Thestar*ngpoint

AnyofthetechniquesbeginwithQualityAssuranceSystemDevelopmethodsforvalida*onValidatemethodsEstablishSOPsforyourqualitycontroltes*ngMaintainassessmentsofsystemsuitability

Manymethodstocharacterizeachemicalwhensufficientmass-mgstograms

Elementalanalysis(CHN)ThermogravimetricanalysisIRInfraredspectroscopyNMRnuclearmagne*cresonanceOp*calspectroscopy(visibleandUV)MassSpectrometryAtomicabsorp*on/ICPX-RayspectroscopyRamanspectroscopyPolarographyGasandLiquidChromatography(usuallyasepara*ontechniqueusedwithabove)electrophoresisO]encansendouttoacompanytoperformthetes*ngalsoImpuri*esWatercontentMel*ngPointOsmolality(solu*on)

Whenthereisonly10mgorless?Dependoninforma*onfrommanufacturer-cer*ficatesofanalysisWhatyouneedtoknow:

Iden%tyPurityAmount/concentra%on

Typicallymanufacturerprovidessomeofthefollowing(2to3)

PuritybyHPLCorGLCo]enIden*ty(purity?)byNMR,someMS,IR,UV,chiralitywhenimportantMel*ngpoint(MP)Elementalanalysis.

Needtoverifyinhouse

Forsynthesis–precursordoesitreacttomakewhatyouwant?Iden%ty:MP(importantforFDG-mannosetriflate)chirality?(difficultwithoutchiralchromatography)/PolarimeterMassspectrometry,UVspectrumandex*nc*oncoefficientcanhelpNMR(takes1+mg)Purity,typicallychromatography(GLC,HPLCorSEC),MP,NMR,ICP,electrophoresis

ValueofanalysisdependsonValidatedEquipmentandMethods

Maintainequipment

Requirementsforamethod

PrecisionAccuracySpecificityRecoveryRuggednessRobustnessStabilityDetermina*on(refs.FDA/COX)

Requirementsforamethod-Precision

Precision“poorlydefined”“Thereproducibilityofmeasurementwithinaset,thatis,tothescaherordispersionofasetaboutitscentralvalue.“Analy*calChemistry(AN75)”(Kateman1993)“Precisionisthemeasureofhowclosethedatavaluesaretoeachotherforanumberofmeasurementsunderthesameanaly*calcondi*onsICHhasdefinedprecisiontocontainthreecomponents:repeatability,intermediateprecisionandreproducibility”Themeanisanes*mateofaccuracyandthe%RSD(rela*vestandarddevia*on)isanes*mateofsampleanalysisprecision(FDAReviewerGuidanceValida*onofChromatographicMethods) Quan*ta*onLimit–Precisionstandarddevia*onatlimitofquan*ta*on

Precisionfunc*on-stddevia*onashavelessandlessanalyteStudenttfactorforNdegreesoffreedom–95%value

LinearRegression

NothingisknownbeyondthemeasuredStandardpointsNeedtobrackettheanalytewithstandardsNeedReplicates,CheckforbiasDetermineuncertain*esintheEs*mateofanalyteconcentra*onfromthelinearregressionofThestandardcurve

LinearRegression–uncertaintyofes*mates

Theuncertaintyofanes*mateofanalyteConcentra*onIsgreaterattheendsoftheLRcurvethanthecenter.Sta*s*csbooksprovideformulastocalculatetheuncertainty

RequirementsforamethodAccuracy

“Theaccuracyofananaly*calprocedureistheclosenessoftestresultsobtainedbythatproceduretothetruevalue”“Theaccuracyofananaly*calprocedureshouldbeestablishedacrossitsrange.”O]encannotjustrunyoursampleagainstonestandardbecauseyouhaveanunknown(ShouldbereportedaspercentrecoveryaccordingtotheFDA)aminimumof3concentra*onsand3replicatesofeachconcentra*onaccordingtotheFDA.Needtobrackettheanalytesignalwithstandardsthataregreaterandlesserconcentra*on.Needreplicatestoassurethatasignalisreal.Is%RSDenough?Forvalida*onofamethodneedtoconsiderbias,uncertainty.Internalstandardscanprovidecorrec*onforinterferencesandrecoveries

Requirementsforamethod-Specificity(Determina%on)

“Theanalyteshouldhavenointerferencefromotherextraneouscomponentsandbewellresolvedfromthem.Arepresenta*veHPLchromatogramorprofileshouldbegeneratedandsubmihedtoshowthattheextraneouspeakseitherbyaddi*onofknowncompoundsorsamplesfromstresstes*ngarebaselineresolvedfromtheparentanalyte.“(FDA)

EET(14,15) 1:10 in ACN

Time2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00

%

2

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00

%

1

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00

%

2

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00

%

2

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00

%

2

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00

%

0

EET111215a12 2: SIR of 3 Channels ES- TIC

1.83e611.25

0.05

EET111215a12 1: Scan ES- TIC

7.45e411.17

1.14 11.75

EET111215a11 1: Scan ES- TIC

8.28e413.021.11 1.70 4.81 6.32

EET111215a10 1: Scan ES- TIC

8.91e413.801.70

EET111215a9 1: Scan ES- TIC

9.05e414.311.72

EET111215a13 1: Scan ES- TIC

6.56e45.201.94 11.449.27 13.99

14,15-epoxy-eicosatrienoicAcidMW320.5

14,15-EETReac*on11,12-EET8,9-EET5,6-EET

Structure-Wikipedia

Below:Separa*onof4of8EETisomers

HPLCwithUV(PDA)andMSSelec*veUVnmSelec*veUVnmMul*plewavelengthsM/ZofFMISORangeofmassesM/ZFMISOM/ZFMISO+Na

Howtostart?Startwithastandardofthecompoundyouaremakingifpossible.Examplefluoromisonidazole(FMISO)

STDFMISO001 D1 C 11.1 ug/mL

Time2.00 4.00 6.00 8.00 10.00 12.00

%

3

2.00 4.00 6.00 8.00 10.00 12.00

AU

0.02.0

2.00 4.00 6.00 8.00 10.00 12.00

AU 0.05.0e-3

2.00 4.00 6.00 8.00 10.00 12.00

AU

0.02.0e-2

FMISO40517a3 5: Diode Array 327

Range: 3.952e-26.68

1.83

FMISO40517a3 5: Diode Array 254

Range: 9.878e-36.68

1.82

FMISO40517a3 5: Diode Array Range: 3.7066.68

1.83

FMISO40517a3 1: SIR of 1 Channel ES+ TIC

3.69e56.84

6.66 STDFMISO001 D1 C 11.1 ug/mL

m/z190 192 194 196 198 200 202 204 206 208 210 212

%

0

100

%

0

100

%

0

100

FMISO40517a3 305 (6.840) 4: Scan ES+ 2.02e4190.01

20224 211.9916896

191.04;1968 211.00;916205.18700

196.91436

FMISO40517a3 307 (6.868) 2: SIR of 1 Channel ES+ 9.49e4190.20

94864

FMISO40517a3 305 (6.821) 1: SIR of 1 Channel ES+ 2.10e5212.20

210176

STDFMISO001 D1 C 11.1 ug/mL

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

%

3

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

%

13

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

%

11

FMISO40517a3 4: Scan ES+ TIC

1.33e56.82

1.681.46 1.86 6.642.86 3.44 4.54 5.97 7.22 9.928.119.39 11.49

FMISO40517a3 2: SIR of 1 Channel ES+ TIC

9.49e46.87

FMISO40517a3 1: SIR of 1 Channel ES+ TIC

3.69e56.84

6.736.66

STDFMISO001 D1 C 11.1 ug/mL

nm200 220 240 260 280 300 320 340 360 380 400

AU

0.0

5.0e-3

1.0e-2

1.5e-2

2.0e-2

2.5e-2

3.0e-2

3.5e-2

FMISO40517a3 395 (6.667) 5: Diode Array 3.572e-2321.88;35716

223.8817109

Symmetricalpeakswithtwodetectortypesgoodindica*onofpurityandiden*ty.Ifdon’thavetwodetectors,trytwoHPLCmethodstodetectimpuri*es.

UVspectrumofFMISO

Requirementsforamethod-Recovery

-100000

0

100000

200000

300000

400000

500000

0 10 20 30 40 50 60 70

Theac*onorprocessofregainingpossesionorcontrolofsomethingstolenorlost”(Google).Thisispartofaccuracy.Usuallywedon’tweightodeterminerecovery.GoodexampleisfluorideonmanyHPLCcolumnswhendeterminingradiochemicalyield.Onsomecolumnssomeorallofunlabeled18F-willberetainedonthecolumnduetosilicabinding.GoodtohaveTLCandHPLCforfluorideradiopharmaceu*cals.Waytotest?Measuretheac*vityinjectedandcollectalloftheeffluentandcomparetoobtainpercentrecovery.Anothermethodformolecules?Internalstandardsoftheauthen*ccompoundbutwhenYoursamplecontainslowmassofmaterial,sta*s*calvaluesmayincreaseuncertain*es.TLCifyoucutandcounthas100%recovery

CutandcountofTLCforFDGreac*on90%rchemyield

18F

FDG

Requirementsforamethod-Robustness,RepeatabilityandReproducibilityRuggednessUSP<1225>isintermediateprecision,robustness,reproducibility

ICHdefinesrobustnessasameasureofthemethod'scapabilitytoremainunaffectedbysmall,butdeliberatevaria*ons·inmethodparameters“(FDA)“Thereproducibility,theclosenessofagreementbetweenindividualresultsobtainedwiththesamemethodandunderdifferentcondi*ons”“Therepeatability,theclosenessofagreementbetweensuccessiveresultsobtainedwiththesamemethodandunderthesamecondi*ons”(Kateman1993)

Reproducibility Repeatability

Above:exampleoftworeadersofpHteststripsgavedifferentresults=poorreproducibility

Above:exampleofrepeatability.GCanalysisofethanol.Somedifferenceinprecisionathigherconcentra*onsbutthesameresultswithintheprecision.Triplereplicatestrianglesareunderneaththesquaresatthelowerconcentra*ons.

Requirementsforamethod-StabilityDoesthesampledegrade(orthedetector)overthe*mecourseofananalysis?Thiscanbeaproblemwithvola*les–example:GC-MSfor4standardsolventscombinedinonevialovera7dayperiod.Theethanolshowsvariability,probablybecausetheconcentra*onIsatthenonlinearresponserangeoftheMS,butnolossofsignalover*me.Acetoneconcentra*onisdecreasingover*me.AcetonitrileandDMSOconcentra*onsarealsochangingwith*me,butatalowerrate.Thesestandardsmustbere-madeeachday.

Requirementsforamethod-PrecisionAccuracyBiasFESHPLCC18RP281nmUVShowsacceptableprecision,accuracyandnosignificantbias.

Compound 1 name: FES(281)Correlation coefficient: r = 0.999983, r 2̂ = 0.999965Calibration curve: 1727.47 * x + 449.253Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

0

3.47e5

Response

0.0 25.0 50.0 75.0 100.0 125.0 150.0 175.0 200.0ug/ml-1.14

1.09

+/- %Conc

Concentration

(µg/mL) Retention

Time (min) Signal Linear

Regression (µg/mL)

200 Mean ± SD (3)

7.4 ± 0.4 7.4 ± 0.9

3.5 ± 0.0 X 105 3.5 ± 0.0 X 105

200 ± 0.6 200 ± 0.9

RSD% 0.60% 0.34% 0.34% 100 Mean ±

SD (3) 7.4 ± 0.3 7.4 ± 0.5

1.7 ± 0.0 X105 1.7 ± 0.0 X105

100 ± 0.6 100 ± 0.9

RSD% 0.57% 0.35% 0.35% 50 Mean ±

SD (3) 7.4 ± 0.3 7.4 ± 0.0

0.87 ± 0.01 X105 0.87 ± 0.00 X105

50 ± 0.6 50 ± 0.9

RSD% 0.43% 0.43% 0.43% 25 Mean ±

SD (3) 7.4 ± 0.1 7.4 ± 0.1

0.35 ± 0.00 X105 0.35 ± 0.00 X105

25 ± 0.6 25 ± 0.9

RSD% 0.31% 0.30% 0.30%

Requirementsforamethod-PrecisionAccuracyBias

FESHPLCC18RP289M/ZMSES-Thesedatashowpoorprecision,nobiasandokayaccuracyfortheconcentra%onwithintheprecisio,butitisnotanacceptableanalysis.

Concentration

(µg/mL) Retention

Time (min) Signal Linear

Regression (µg/mL)

2.0 Mean ± SD (3)

7.8 ± 0.2 7.6 ± 0.0

8.7 ± 0.8 X 106 6.5 ± 0.4 X 106

2.3 ± 0.2 1.6 ± 0.0

RSD% 0.89% 18% 21% 1.0 Mean ±

SD (3) 7.7 ± 0.0 7.6 ± 0.0

6.2 ± 1.6 X106 3.6 ± 0.6 X106

1.5 ± 0.0 0.7 ± 0.0

RSD% 0.54% 29% 39% 0.50 Mean ±

SD (3) 7.7 ± 0.0 7.6 ± 0.0

3.8 ± 0.5 X106 2.1 ± 0.6 X106

0.8 ± 0.0 0.25 ± 0.02

RSD% 0.36% 32% 56% 0.25 Mean ±

SD (3) 7.6 ± 0.1 7.6 ± 0.1

2.3 ± 0.5 X106 0.3 ± 0.4 X106

0.32 ± 0.01 0.01 ± 0.01

RSD% 0.3% 31% 103%

Compound 1 name: FES(289.4)Correlation coefficient: r = 0.900260, r 2̂ = 0.810469Calibration curve: 3.26640e6 * x + 1.26295e6Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

0

9.65e6

Response

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0ug/ml-95.6

59.8

+/- %Conc

Requirementsforamethod-PrecisionAccuracyBiasFESHPLCC18RP289M/ZMSES-FESHPLCC18RP281nMUVLinearregressionMS:r2=0.998UV:r2=0.983Varia%onofstandardconcfromcurveto10%to25%Es%matedanalyteconc:0.21±0.010.25±0.08µg/mLUVisnearminimumquan%ta%onlimit.MSisadetec%onmethodwithchangingsensi%vity,internalstandardscanhelpaccountforchangesindetectorsensi%vity.

Compound name: FES 289Correlation coefficient: r = 0.999146, r 2̂ = 0.998294Calibration curve: 514064 * x + 6705.55Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

ug/mL0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800Re

spon

se

0200000400000

ug/mLResid

ual

-10.00.0

Compound name: FES uvCorrelation coefficient: r = 0.991330, r 2̂ = 0.982735Calibration curve: 195.762 * x + 15.7048Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

ug/mL0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800Re

spon

se

0100

ug/mLResid

ual

-25.00.0

Chromatography

•  Columnorplate(sta*onaryphase)passaliquidthroughitandtherela*veaffinityfortheanalyteastheliquid(orgas)passesthroughhasitretainedonthecolumnthenbackintosolu*onforrela*velydifferent*mes.Themoretheanalyteisinterac*ngwiththesta*onaryphasethelongeritisretainedonthecolumn.

•  Concepts-efficiency(theore*calplates) -Deadvolume– -resolu*on(peaksepara*on) -Capacity(amountofmassthatcangoontothecolumn) -peaksymmetry/tailingfactor.

•  ASEP-PAKcanbeusedforchromatographybutisalmostalwaysabinaryevent…onoroffandtheaffinityfortheliquidthatpassethroughdeterminesonoroff.

•  TwoCrucialconcepts–  Limitofdetec*on–  Limitofquan*ta*on

SystemSuitability-Sensi*vitySensi*vityiswhatthelowerlimitofabilitytoassessinvolvesthewholesystem•  Twoimportantconcepts

–  Limitofdetec*on~3*messtandarddevia*onofpeaktopeaknoise(S/N?)

–  Limitofquan*ta*on~10*messtandarddevia*onofpeaktopeaknoise(S/N?)Ref(Kateman1993)

MBRFES005A

Time2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

54

2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

AU

-2.0e-1

0.0

2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

AU

0.0

2.5e-4

5.0e-4

FES060717a11 5: Diode Array 280

Range: 7.8e-43.03 5.83

3.434.02

5.274.18 5.02 7.787.686.15 6.62

FES060717a11 5: Diode Array Range: 4.933e-12.72 5.85

3.275.23 7.82

FES060717a11 1: SIR of 1 Channel ES- TIC

2.72e46.01

2.70 5.655.384.954.453.996.28

7.21 7.45

MBRFES005A

Time2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

54

2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

AU

-2.0e-1

0.0

2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

AU

0.0

2.5e-4

5.0e-4

FES060717a11 5: Diode Array 280

Range: 7.8e-43.03 5.83

3.434.02

5.274.18 5.02 7.787.686.15 6.62

FES060717a11 5: Diode Array Range: 4.933e-12.72 5.85

3.275.23 7.82

FES060717a11 1: SIR of 1 Channel ES- TIC

2.72e46.01

2.70 5.655.384.954.453.996.28

7.21 7.45

Impuri*es

MBRFMISO-W0023

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

%

50

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

AU

-1.0e-2

0.0

1.0e-2

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50

AU

-5.0e-3

0.0

5.0e-3

fmiso020410a10 Sm (Mn, 2x4) 5: Diode Array 327

Range: 1.465e-2Area

5.171105

4.10174

8.50768

9.40751

fmiso020410a10 5: Diode Array 327

Range: 3.004e-21.72

1.37

2.005.172.67 8.50 9.40

fmiso020410a10 1: SIR of 1 Channel ES+ TIC

3.05e48.64

8.58

8.50

1.450.52 0.69

1.56 2.142.73 5.342.84 3.71 3.97 5.05 7.345.89 7.106.35 7.77

8.70

8.90

9.54

20uLinj 10uLinjRet*mePkarea ug/mL ug/mL

4.1 174 0.106 0.0535.17 1105 0.818 0.418.5 768 0.560 0.28 FMISO9.4 751 0.547 0.27

Totalimpuri*es: 0.74ug/mLAretheseallimpuri*esintheproduct?ThisisaUVchromatogramwiththepeaksintegrated.UVsignalisontheYaxis,elu*on*meinminutesisontheXaxis.Theproductisat8.5minutes.Peaksbefore2.8minutesarefromthesolventfront.Thepeakintegratedat4.1minutesisbelowtheminimumquan*ta*onlimit(eventhoughtheso]warequan*tatedit).Thepeakat5.2minutesIsaknownimpurity.Thepeakat9.40minutesisintheHPLCsolvent.Thustherealquan*fiableimpurityisthepeakat5.2minutes.Wemaketheassump*onofconcentra*onbasedonthesamesensi*vityastheFMISOforUV.Thisisanassump*on.

Valida*onbeyondtypicalanalysis:“Effec*veSpecificRadioc*vity”ismeasuredconcentra*onreal?Thefiguresshowsareceptorbindingassaycomparedwiththemeasurementofmassoffluoroestradiol(FES)fromHPLfor5batchesofFES.Thetestshowedthatthebiologicalbindingcomparedwithmeasuredmassagreedwithexpectedbindingforfluoroestradiol(FES),whichisslightlylowerthanthena*veligand,17β-estradiol

Requirementsforamethod-Recovery

Puritychecks-Howradiochemicallypureisyour18Fradiopharmaceu*cal?Whatwasyourradiochemicalyield?HPLC98%butTLC70%TLChas~100%recoveryforcoun*ng.YoucountthewholesampleHPLCcanhavefluoridesorbontothesta*onaryphaseandnevercomeout.Typicallythisisdeterminedduringmethodvalida*onbyinjec*ngameasuredamountofanalytebeforetes*ng(e.g.beforeinjec*ngoncolumn)andthenmeasuringwhatisrecoveredfromtheanalytepeakandcomparingtheAmountinjectedwiththeamountrecovered.Foranalyterecovery,internalstandardsareo]enusedthatarenotthesamemoleculeastheanalyteandareco-injectedwiththeanalyte,butassump*onsthatrecoveryofanalyteandstandardareequalhavetobeproven.Injec*onofInternalstandardsofthesamemoleculeastheanalytearebest.Thisisstandardaddi*on.Howeverattracelevelsrecoverymaynotbethesame.

SystemSuitability•  Forchromatographysystemsuitabilityisessen*al•  Assuresforachromatographyanalysis:

–  Sensi*vity-0.1%fordrugproduct,0.05%drugsubstance–  RefUSP<621>chromatography

–  Fora4µglimitinjec*ondose20mLmeansconcneedstobe4µg/20mL=0.69pmol/mLsoifat0.1%ofthatvalueisasensi*vityof0.69fmol/mLforLOQExtremetlydifficultespeciallyinamatrixofUSPsolvents(water/ethanol)bestprac*cescanbethebestyoucando

–  Needtohavesensi*vityandaccuracyatorbelowlimitallowable–  Ifthemeasureassuresthatyouaresta*s*callyabletoshowpurity,

accuracyandthatyouarebelowlimitsofadministra*onwithsta*s*calsignificance,thenthatisenough.

SystemSuitability-•  Showthatthemethodscon*nuetoworkthatdayandover*me

•  Peaksymmetry,tailingfactor,theore*calplates

•  Checksaccuracy,sensi*vity,precision,ruggedness

Sample Retention time 1750 µg/mL isopropanol

(min)

Retention time 2500 µg/mL ethanol (min)

Resolution Peak Tailing Isopropanol /

ethanol

Capacity Factor isopropanol /

ethanol Theoretical Plates

Referenceswww.agilent.com“Valida*onofAnaly*calTechniques”LudwigHuberaprimerhhps://www.fda.gov/downloads/drugs/guidances/ucm073384.pdfGuidanceforIndustryQ2BValida*onofAnaly*calProcedures:MethodologyNov1996GuidanceforIndustryQualitySystemsApproachtoPharmaceu*calcGMPRegula*onsSept2006USDeptofHealthandHumanServicesFoodandDrugAdministra*on.CEDRCEBRNov1996USP40<1225>Valida*onofCompendialProceduresUSP40<621>Chromatography,<736>MassSpectrometry,<821>radioac*vityICHInterna*onalCommiheeonHarmoniza*on-mul*pledocumentsZarBiosta*s*calAnalysis(anygoodsta*s*csbookdealingwithregressionanalysisGKateman,LBuydens,JDWinefordner(SeriesEditor)QualityControlinAnaly*calChemistry2ndEdi*on.JWiley&Sons.1993.SCox.Pharmaceu*calManufacturingHandbook.Regula*onsandQuality.WileyInterscience(online)