08/07/2017 A Method for the Qualitative and Quantitative Determination of the Amino Acid Composition of Pharmaceutical Products | SGS

AMETHODFORTHEQUALITATIVEAND
QUANTITATIVEDETERMINATIONOFTHE
AMINOACIDCOMPOSITIONOF
PHARMACEUTICALPRODUCTS
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June24,2016

Author:

NataliaBelikova,Ph.D.,AnalyticalServicesDirectorLifeScience,Lincolnshire,IL,USA

TheUnitedStatesPharmacopeialConvention(USP),EuropeanPharmacopoeia(Ph.Eur.)
andJapanesePharmacopoeia(JP)definerequirementsforthequalitativeandquantitative
compositionofmedicines.Thesepharmacopeiasalsooutlinetheanalyticalmethodstobe
carriedoutonpharmaceuticalproductsaswellasthesubstancesandmaterialsusedin
theirproduction.

AminoAcidAnalysisingeneralcanbeusedforidentificationtestingofbiopharmaceutical
activeingredientsandthedeterminationofimpuritiesandrelatedsubstancesinactive
pharmaceuticalingredients.Itcanalsobeusedforsingleortotalaminoacidquantitationin
drugproducts.Ourobjectivewastodevelopaliquidchromatographymethodfor
simultaneousdeterminationofapproximately20aminoacidsinsimpleandcomplex
mixturesthatcomplywiththesystemsuitabilityrequirementsofthePh.Eur.general
chapter(2.2.56).

Applications

Formethoddevelopment,theworkingstandardcontained17differentaminoacidsat
concentration25pmol/L(AgilentTechnologies)wasusedtoevaluateseparationand
reproducibility.TheprogramwasoptimizedtohaveresolutionbetweenLeucineand
Isoleucineatleast1.5.asperPh.Eur.generalchapter2.2.56requirements.Also,ammonia
standard(0.12ppmor0.02%relativelytothesampleconcentration)wasrunatthesame
timetoshowthatpeakdoesnotinterferewithotherpeaksandhavesufficientpeakarea
countstobecalculatepreciselyfromruntorun.Ph.Eur.monographsforArginineand
Histidine(ninhydrinpositivesubstancesandammoniumtests)wereofficiallyverifiedatour
location.Severalothermonographs,includingLLysine,LArginine,LGlycinewere
successfullyruninourlab.

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08/07/2017 A Method for the Qualitative and Quantitative Determination of the Amino Acid Composition of Pharmaceutical Products | SGS
Lateron,HPLCmethodwithpostcolumnderivatizationtotestninhydrinpositive
substancesandammoniuminLArginineperPh.Eur.hasbeenverified.Monographlisted
LOrnithine,LLysineandLCitrullineasknownimpurities.ValidationincludedSpecificity,
Linearity,LOQ,LOD,Precision,IntermediatePrecisionandAccuracy(spikerecovery).The
methodhasbeenshowntobespecific,linear,accurateandprecise.

SGSLifeSciences(Chicago,IL)successfullyappliedthismethodtotestaminoacids
presentincomplexformulations.Forexample,ArginineHydrochlorideservesasstabilizer
forabiosimilarproteinbasedantiinflammatorydrug.ThehighmolecularweightAPIwas
filteredoutandalowmolecularweightfractionwasdilutedandinjectedforanalysis.We
demonstratedexcellentlinearity(correlation0.9997atrange0.5100g/mL),goodmethod
precision(%RSDn=5=0.20.6%)andacceptableaccuracyacrossthe75125%range
(recoverywithin96121%)whenperformedspikerecoverystudyusingPlacebo.

Atlast,methodisbeingusedroutinelytoanalyzeaminoacidcompositionofsmallproteins
andpolypeptides.Sampleishydrolyzedwith6NHClat110Cforatleast20hoursandthen
injected.Individualaminoacidsareidentifiedandquantitatedagainstknownstandards.
Thistestservesasanidentificationtestandisbasedonindividualaminoacidrelative
composition(relativeproportionofeachaminoacidtothetotalaminoacidpresentinthe
sample).

Method

AnHPLCSystemwithasolventdeliverysystem,anautosampler,adualwavelength
detectoranddatacollectionmodule(Waters)wereused.Themethodutilizedamobile
phasegradientprogramaswellasacolumntemperaturegradientprogramforseparation
ofaminoacidsofinterest.Afterseparation,eluentunderwentpostcolumnninhydrin
derivatizationwithsubsequentdetectionat570nmand440nmforaminoandiminoacids,
respectively(PinnaclePCXPostColumnDerivatizationInstrument).TheHPLCparameters
areoutlinedinTable1andthemobilephasegradientprogramispresentedinTable2.
Table3illustratesthepostcolumnderivatizationsystemparametersandthecolumn
temperaturegradientprogramispresentedinTable4.

ThestandardsandtestsamplewereinjectedintoanHPLCwithasodiumbasedcolumn
(PickeringLabsSodiumExchange,4.6110mm,5m).Thesampleswereseparatedby
amobilephasegradientprogramcoupledwithatemperaturegradientprogramtooptimize
aminoacidseparation.Thecolumneluentunderwentpostcolumnderivatizationwith
ninhydrin.Ninhydrinpositivesubstancesbasedonaminoacidsweredetectedat570nm
ninhydrinpositivesubstancesbasedoniminoacids,suchasprolineweredetectedat440
nm.

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08/07/2017 A Method for the Qualitative and Quantitative Determination of the Amino Acid Composition of Pharmaceutical Products | SGS

Results

UsingthePh.Eur.generalchapter(2.2.56)method,wewereabletoseparateandidentify
theaminoacids.Noblankinterferencewasobservedattheretentiontimeofaminoacidsof
interest,indicatingthespecificityofthemethod(Figure1).Blankpeaksduetothemobile
phasegradientwereassignedbasedonblankinjection.Likewise,theammoniumstandard
gaveasinglepeak(Figure2).Theaminoacidsinthestandardmixdemonstratedgood
separationandwereeasilyidentifiable(Figure3),asdidthehydrolysedtestsample(Figure
4).Proline,whichisaniminoacid,wastheonlypeakdetectedat440nm(Figure5).

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Theassaywastestedforsuitabilitywithasetofselectaminoacids(Table5).The%RSDof
theworkingstandard(instrumentprecision)foraminoacids(~1.2g/mLor0.2%tothe
sampleconcentrationforanalysisofindividualaminoacidsperPh.Eur.monographs)was
notmorethan15%.The%RSDforammoniumpeak(0.12ppmor0.02%tothesample
concentration)wasnotmorethan15%.ResolutionbetweenLeucineandIsoleucinewas
notlessthan1.5.Peaksymmetryforaminoacidsandammoniumpeakwas0.81.5.The
methodhasbeenshowntobelinearforselectedaminoacidsintherangeof0.31.5g/mL
(Table6).TheLOQofthemethodforseveralaminoacidswas~0.3g/mLor0.05%
relativetothesampleconcentrationwhereas,theLODofthemethodwas~0.1g/mLor
0.015%relativetothesampleconcentration.

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Conclusions

Thedevelopedmethodwasoptimizedtocomplywithsystemsuitabilityrequirementsper
thePh.Eur.generalchapter(2.2.56).Ourlaboratoryhassuccessfullyverifiedthemethod
foranalysisofninhydrinpositivesubstancesinindividualaminoacidsamplesperthePh.
Eur.monographs.Whilethismethodhasbeenusedtoevaluateexcipients(aminoacids)
concentrationsingenericandbiosimilardrugs,themethodhasbeenusedtoevaluate
relativeaminoacidcompositioninsmallproteinsandpeptides.

References

Ph.Eur.9.0(2.2.56)AminoAcidAnalysis

Ph.Eur.8.0ArginineHydrochlorideMonograph

USP39<1226>VerificationofCompendialProcedures

PickeringLaboratoriesMethodAbstract/391AminoAcidAnalysisaccordingtoEuropean
Pharmacopeia8.0

ICHQ2(R1)ValidationofAnalyticalProcedures,TextandMethodology

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