BRIEFING

858RamanSpectroscopy,PF40(6)[Nov.Dec.2014].Thisrevisionisbeingproposedto
addressthecommentsreceived.Inthisproposal,theCommitteehasmadeaneffortto
harmonizewiththeEuropeanPharmacopoeia,totheextentpossible.USPhasreceiveda
numberofcommentsrequestingguidanceonmultivariatecalibrationssincethechapter
focusesontheunivariateapproachonly.AnewchapteronChemometrics1039hasbeen
proposedinPF41(6),andsincethischapterisstillinthecommentperiod,itcannotbe
referencedinanyofthespectroscopychapters.Once1039isofficial,thenonecanuseitfor
guidanceasnecessary.
Additionally,minoreditorialchangeshavebeenmadetoupdatethechaptertoUSPstyle.
(GCCA:K.Zaidi.)
CorrespondenceNumberC157780
Commentdeadline:May31,2016
Addthefollowing:
858RAMANSPECTROSCOPY
Changetoread:
Ramanisavibrationalspectroscopictechniqueandisthereforerelated complementary 1S(
USP40)
toinfrared(IR)andnearinfrared(NIR)spectroscopy.TheRamaneffectitselfarisesasa
resultofachangeinthepolarizabilityofmolecularbondsduringagivenvibrationalmodeand
ismeasuredasinelasticallyscatteredradiation.TheappearanceofaRamanspectrumismuch
likethatofanIRabsorbancespectrum.Theintensities,orthenumberofRamanphotons
counted,areplottedagainsttheshiftedenergies.TheRamanshiftisusuallyexpressedin
wavenumberandrepresentstheenergydifferencebetweentheincidentphotonenergyandthe
inelasticallyscatteredphotonenergy.UnlikeIRorNIRspectrathatarisewhenvibrational
modesinamoleculehaveadipolechange,Ramanspectrahavefrequenciesandintensities
thatarisewhenthesesamemodescauseachangeinpolarization polarizability. 1S(USP40)
Thespectrumisinterpretedinthesamemannerasthecorrespondingmidinfraredspectrum.
Thepositionsofthe(Ramanshifted)wavenumbersforagivenvibrationalmodeare
theoretically 1S(USP40)
identicaltothewavenumbersofthecorrespondingbandsinanIRabsorptionspectrum.
However,thestrongerpeaksinaRamanspectrumareoftenweak ,ornotpresent 1S(USP40)
inanIRspectrum,andviceversa.Similarly,wavenumbersabove1500cm1aregroup
frequencies,andstrongbandsthatabsorbbelow1500cm1canbeeithergroupfrequencies
orfingerprintbandsforthemolecule.Thus,thetwospectroscopictechniquesareoftensaidto
becomplementary.Fordiscussionofthetheoryandprinciplesofmeasurements,seeRaman
Spectroscopy1120,whichmaybeahelpful,butnotamandatory,resource.
Changetoread:
QUALIFICATIONOFRAMANSPECTROMETERS
QualificationofRamanspectrometersisdividedintothreecomponents:1)installation
qualification(IQ)2)operationalqualification(OQ)and3)performancequalification(PQ).For
furtherdiscussion,seethegeneralinformationchapterAnalyticalInstrumentQualification

1058.
InstallationQualification
TheIQrequirementsprovideevidencethatthehardwareandsoftwareareproperlyinstalled
inthedesiredlocation.
OperationalQualification
Acceptancecriteriagiveninthissectionareapplicableforgeneraluse.Acceptancecriteria
forparticularinstrumentsandapplicationscanvary,dependingontheanalyticalmethodused
andthedesiredaccuracyofthefinalresult.ASTMstandardreferencematerialsarealso
specified,withtheunderstandingthatinsomecircumstances(specifically,remoteonline
applications),calibrationusingoneofthesematerialsmaybeimpracticalandothersuitably
verifiedmaterialscanbeused.
WAVELENGTHACCURACY
Theaccuracyofthewavelengthaxisisensuredviacalibrationtomaintaintheintegrityof
Ramanpeakpositions.WavelengthcalibrationofaRamanspectrometerconsistsoftwoparts:
primarywavelengthaxiscalibrationandlaserwavelengthcalibration.Afterboththeprimary
wavelengthaxisandthelaserwavelengtharecalibrated,instrumentwavelengthuncertainty
canbedetermined.Theinstrumentwavelengthuncertaintycanbedeterminedbyusinga
Ramanshiftstandardorothersuitablyhighpuritymaterial(e.g., Acetaminophen, 1S(USP40)
Cyclohexane,orpolystyrene).Selectionofastandardwithbandspresentacrossthefull
Ramanspectralrangeisrecommendedsothatinstrumentwavelengthuncertaintycanbe
evaluatedatmultiplelocationswithinthespectrum.Usingasuitable,wellcharacterized
wavenumberstandard,theanalystconductsthecalibrationscansfrom3800to350cm1 over
thespectralrangeoftheanalyticalsystem 1S(USP40)
wavenumbersandcomparesthewavenumberofmaximumresponseofseveralchosenbands
usingthecenterofgravity,polynomialsplineprocedure,orotherpeakpickingalgorithmsto
theknownRamanshiftofthestandard(seeTable1).
Table1.RecommendedWavelengthPeakValuesforCyclohexane,Naphthalene,and
Polystyrene(cm1)
Cyclohexane

Naphthalene

Polystyrene

384.1

513.8

620.9

426.3

763.8

795.8

801.3

1021.6

1001.4

1028.3

1147.2

1031.8

1157.6

1382.2

1155.3

1266.4

1464.5

1450.5

1444.4

1576.6

1583.1

2664.4

3056.4

1602.3

2852.9

2852.4

2923.8

2904.5

2938.3

3054.3

Table1.WavenumberShifts(andAcceptableTolerances)ofPolystyrene,
Acetaminophen,andCyclohexane
WavenumberShiftsa

TolerancesBenchtop

(cm1)

Polystyreneb

(cm1)

620.9

1.5

1001.4

1.5

1031.8

1.5

1602.3

1.5

3054.3

3.0

Acetaminophen

797.2

2.5

857.9

1.5

1168.5

1.5

1236.8

1.5

1323.9

1.5

1648.4

1.5

2931.1

2.0

801.3

1.5

1028.3

1.0

1266.4

1.0

1444.4

1.0

2852.9

2.0

Cyclohexane

a StandardguideforRamanshiftstandardsforspectrometercalibration(American
SocietyforTestingandMaterialsASTME1840).
b Polystyrenefoil(30mil,76micron)orPolystyrenepellets(NIST706a).
c AcetoaminophenforequipmentqualificationCRS.
d CyclohexaneR.
1S(USP40)

Acceptancecriteriaforwavelengthaccuracyare1.5cm1oftherecommendedvalues.
[NOTEForscanningdispersiveinstruments,calibrationmightneedtobeperformedmore
frequently,andprecisioninbothascanningandstaticoperationmodemayneedtobe
verified.]
PHOTOMETRICPRECISION
Laservariationintermsofthetotalemittedphotonsoccurringbetweentwomeasurements
cangiverisetochangesinthephotometricprecisionoftheinstrument.Unfortunately,itis
verydifficulttoseparatechangesinthephotometricresponseassociatedwithvariationsinthe
totalemittedlaserphotonsfromthesampleandsamplinginducedperturbations.Thisisone
ofthereasonswhythephotometricprecisionacceptancecriteriaaresetrelativelylooselya
toleranceof10%fromreferencemeasurementsmadefromreferencematerialisapplied.
PerformanceQualification
Theobjectiveofperformancequalificationistoensurethattheinstrumentisperforming
withinspecifiedlimitswithrespecttowavelengthandphotometricprecision.Incertaincases,
whentheinstrumenthasbeensetupforaspecificmeasurement,itmightnolongerbe
possibleordesirabletomeasurethewavelengthandphotometric(signal)qualification
referencestandardsusedintheOQ.ProvidedthatinstrumentOQhasshownthatthesystemis
fitforuse,asingleexternalperformanceverificationstandardcanbeusedonacontinuing
basis(e.g.,dailyorbeforeuse).Theperformanceverificationstandardmustmatchthe

formatofthesamplesinthecurrentanalysisascloselyaspossibleandmustusesimilar
spectralacquisitionparameters.Quantitativemeasurementsofanexternalperformance
verificationstandardcanbeusedtocheckboththewavelengthaccuracyandthephotometric
precision.

PROCEDURE
Ramanspectracanbeobtainedfromseveralsamplepresentationsincludingsolids,
powders,slurries,gels,liquids,films,andgases.Inaddition,measurementcanbeperformed
throughglassorplasticfilmsthatarenormallyusedforcontainment.Typically,Raman
spectroscopydoesnotrequireanysamplepreparationbecausethedetectorshaveawide
dynamicrangeandthecollectiontimecanbeadjustedbytheuser.Considerationsabout
exposureofthesampletothelasermustbetakenintoaccountbecausehighintensitylasers
andsmallmeasurementspotsmayresultinburningofthesample.
Itisimportanttonotethatspecificparameters,suchasspectrometernoise,limitsof
detection(LOD),limitsofquantification(LOQ),andacceptablespectralbandwidthforany
givenapplicationmustbeincludedaspartoftheanalyticalprocedure.Specificvaluesfortests
suchasspectrometernoiseandbandwidthwilldependontheinstrumentchosenandthe
requiredpurpose.Forthisreason,specificinstrumenttestsfortheseparametersarenot
indicatedhere.Foradditionaldetails,seegeneralinformationchapter1120,whichprovidesa
detaileddiscussionofRamanspectroscopy.
Changetoread:
VALIDATIONANDVERIFICATION
TheobjectiveofRamanprocedurevalidation,asisthecasewithvalidationofanyanalytical
process,istodemonstratethatthemeasurementissuitableforitsintendedpurpose.The
procedureforvalidationisrelatedtothefundamentalvalidationcharacteristicsrequiredfor
anyanalyticalprocedure.Datapretreatmentisoftenavitalstepinthechemometricanalysis
ofRamanspectraldata.Manysuitabledatapretreatmentsexisttheselectionshouldbebased
onsoundscientificjudgmentandsuitabilityfortheintendedapplication.

Validation
ValidationisrequiredwhenaRamanprocedureisintendedforuseasanalternativetothe
officialprocedurefortestinganofficialarticle.TheobjectiveofaRamanprocedurevalidation
istodemonstratethatthemeasurementissuitableforitsintendedpurpose,including:
quantitativedeterminationofthemaincomponentinadrugsubstanceoradrugproduct
(CategoryIassays)quantitativedeterminationofimpuritiesorlimittests(CategoryII)and
identificationtests(CategoryIV)seeValidationofCompendialProcedures1225,Table2.
Dependingonthecategoryofthetest,theprocessforanalyticalprocedurevalidationfor
Ramanspectroscopyrequiresthetestingoflinearity,range,accuracy,specificity,precision,
detectionlimit,quantitationlimit,androbustness.ValidatedRamanproceduresmaynotbe
transferrabletoallmodelsandconfigurationsofRamanspectrometersbecauseofdifferences
intheirinherentperformancecharacteristics.
Chapter1225providesdefinitionsandgeneralguidanceonanalyticalproceduresvalidation
withoutindicatingspecificvalidationcriteriaforeachcharacteristic.Thesectionsthatfollow
areintendedtoprovidetheuserwithspecificvalidationcriteriathatrepresenttheminimum

expectationsforthistechnology,assumingthetypicalCategoryIUSPacceptancecriteriaof
98.0%102.0%foradrugsubstanceand90.0%110.0%foradrugproduct.Theactual
validationperformancecharacteristicswoulddependontheacceptancecriteriainplaceand
mustprovidesufficientevidencethatthemeasurementcapabilityissufficientforthese
acceptancecriteria.Foreachparticularapplication,tightercriteriamaybeneededto
demonstratesuitabilityfortheintendeduse.
Accuracy
ForCategoryIandIIprocedures,accuracycanbedeterminedbyconductingrecovery
studieswiththeappropriatematrixspikedwithknownconcentrationsoftheanalyte.
ComparisonofassayresultsobtainedusingtheRamanprocedureundervalidationwiththose
obtainedfromanestablishedalternativeanalyticalproceduremayalsobeusedtodetermine
accuracy.
Validationcriteria:98.0%102.0%recoveryfordrugsubstances,95.0%105.0%recovery
fordrugproductassays,and70.0%150.0%recoveryforimpurityanalyses.Thesecriteria
shouldbemetthroughouttheintendedrange.
Precision
REPEATABILITY
Theanalyticalprocedurecanbeassessedbymeasuringtheconcentrationsofsix
independentlypreparedsamplepreparationsat100%oftheassaytestconcentration.
Alternatively,thisassessmentcanbebasedonmeasurementsofthreereplicatesofthree
separatesamplesolutionsatdifferentconcentrations.Thethreeconcentrationsshouldbe
closeenoughthattherepeatabilityisconstantacrosstheconcentrationrange.Ifthisisdone,
therepeatabilityatthethreeconcentrationsispooledforcomparisontotheacceptance
criteria.
Validationcriteria:TherelativestandarddeviationisNMT1.0%foradrugsubstance,NMT
2.0%foradrugproduct,andNMT20.0%foranimpurityanalysis.
INTERMEDIATEPRECISION
Analyststesttheeffectonanalyticalprecisionofchangesinvariables,suchasperforming
theanalysisondifferentdays,usingdifferentinstrumentation,orhavingtheprocedure
performedbytwoormoreanalysts.Ataminimum,anycombinationofatleasttwoofthese
factors,totalingsixexperiments,willprovideanestimateofintermediateprecision.
Validationcriteria:TherelativestandarddeviationisNMT1.0%foradrugsubstance,NMT
3.0%foradrugproductassay,andNMT25.0%foranimpurityanalysis.
Specificity
ForCategoryIandIIprocedures,thespecificityisdemonstratedbymeetingtheaccuracy
requirements.ForCategoryIVprocedures,theidentityoftheanalytemustbeconfirmedby
comparisonwithappropriatereferencesubstances. Suitablechallengetestingshouldbe
undertakentoverifySpecificitybytheuseofanIndexMatchtoanappropriateand
challengingspectrallibrary. 1S(USP40)
QuantitationLimit
Ameasurementofarepresentativesamplematrixspikedattheestimatedquantitationlimit
(QL)concentrationmustbeperformedtoconfirmaccuracy.TheQLcanbeestimatedby

calculatingthestandarddeviationofNLTsixreplicatemeasurementsofablanksolutionand
multiplyingby10.Alternatively,thestandarddeviationcanbedeterminedfromtheerrorof
theinterceptfromacalibrationcurveorbydeterminingthatthesignaltonoiseratiois>10.
Othersuitableapproachescanbeused(see1225).
Ameasurementofatestsolutionpreparedfromarepresentativesamplematrixspikedat
therequiredQLconcentrationmustbeperformedtoconfirmsufficientsensitivityand
adequateprecision.TheobservedsignaltonoiseratioattherequiredQLmustbe>10.
ForCategoryIIprocedures,theanalyticalproceduremustbecapableofdeterminingthe
analytepreciselyandaccuratelyatalevelequivalentto50%oftheacceptancecriteria.
Validationcriteria:FortheestimatedQLtobeconsideredvalid,themeasuredconcentration
mustbeaccurateandpreciseatalevel50%oftheacceptancecriteria.
Linearity
AlthoughRamanspectroscopydoesnotobeytheBeerLambertLaw,Ramanintensityis
directlyproportionaltotheconcentrationofthescatteringspecies.Alinearrelationship
betweentheanalyteconcentrationandtheRamanspectrumresponsemustbedemonstrated
bypreparingNLTfivestandardpreparationsatconcentrationsencompassingtheanticipated
concentrationrangeofthetestpreparation.Thestandardcurvemustbeevaluatedusing
appropriatestatisticalmethods,suchasaleastsquaresregression.
Validationcriteria:Forthecorrelationcoefficient(R),NLT0.995forCategoryIassaysand
NLT0.99forCategoryIIquantitativetests
Range
Thisparameterisdemonstratedbymeetinglinearity,precision,andaccuracyrequirements.
Validationcriteria:ForCategoryItests,thevalidationrangefor100.0%centeredacceptance
criteriais80.0%120.0%.Fornoncenteredacceptancecriteria,thevalidationrangeis10.0%
belowthelowerlimitto10.0%abovetheupperlimit.Forcontentuniformity,therangeis
70.0%130.0%.ForCategoryIItests,thevalidationrangecovers50.0%120.0%ofthe
acceptancecriteria.
Robustness
Thereliabilityofananalyticalmeasurementmustbedemonstratedbymakingdeliberate
changestoexperimentalparameters.ForRamanspectroscopy,thiscanincludebutisnot
limitedtochangesinsampleposition,laser/sampleinteraction,orhardwaresettings.

VERIFICATION
U.S.CurrentGoodManufacturingPracticeregulations[21CFR211.194(a)(2)]indicatethat
usersofanalyticalproceduresdescribedinUSPNFarenotrequiredtovalidatethese
proceduresifprovidedinamonograph.Instead,theusersmustverifysuitabilityunderactual
conditionsofuse.
TheobjectiveofaRamanspectroscopicprocedureverificationistodemonstratethatthe
procedure,asprescribedinspecificmonographs,isbeingexecutedwithsuitableaccuracy,
sensitivity,andprecision.VerificationofCompendialProcedures1226notesthatifthe
verificationofthecompendialprocedure,accordingtothemonograph,isnotsuccessful,the
proceduremaynotbesuitableforusewiththearticleundertest.Itmaybenecessaryto

developandvalidateanalternativeprocedureasallowedinGeneralNoticesandRequirements
6.30TestingPracticesandProcedures,AlternativeandHarmonizedMethodsandProcedures.
Althoughcompleterevalidationofacompendialprocedureisnotrequired,verificationofthe
compendialRamanspectroscopicprocedureincludesthedemonstrationofcertainvalidation
parameters.Whentheprocedurebeingverifiedisforidentificationpurposes,specificityisthe
onlyparameterrequired.However,forquantitativeapplications,additionalvalidation
parametersmustbestudiedasappropriatethesemayincludeaccuracy,precision,andQL,as
indicatedinValidation. 1S(USP40)
AuxiliaryInformationPleasecheckforyourquestionintheFAQsbeforecontactingUSP.