6/30/2016

40(5)InProcessRevision:AztecMarigoldZeaxanthinExtract

BRIEFING
AztecMarigoldZeaxanthinExtract,page6525oftheFirstSupplementtoUSP37andPF39(3)
[MayJune2013].Onthebasisofcommentsreceived,thefollowingchangesareproposed:
1.CorrectatypoontheLatinbinomialintheDefinition.
2.ChangethelimitforOtherrelatedcompoundsinthetestforLuteinandOtherRelated
CompoundsfromNMT1.0%toNMT2.0%
3.ReplacetheImpuritiescategorywiththeContaminantscategory.
4.ReplacetheHeavyMetals 231 testwiththeElementalImpurities 233 test.
5.DeletethetestforResidueIgnition 281 becauseitisnotneededforabotanicalextract.
(DS:H.Dinh.)
CorrespondenceNumberC148522
Commentdeadline:November30,2014
Addthefollowing:
AztecMarigoldZeaxanthinExtract

C40H56O2568.87
(allE)1,1'(3,7,12,16Tetramethyl1,3,5,7,9,11,13,15,17octadecanonaene1,18diyl)bis[2,6,6
trimethylcyclohexene3ol]
3R,3R , Carotene3,3diol[148683].
DEFINITION
Changetoread:
AztecMarigoldZeaxanthinExtractisapurifiedextract,derivedfromtheflowersofTargetes
Tagetes 2S(USP38)
erectaL.,grownfromseedsofvarietiesoftheScarletadecultivarrichinzeaxanthin.Theextract
containsNLT36.0%oftotalcarotenoidscalculatedaszeaxanthin(C40H56O2),NLT30.0%ofall
transzeaxanthin,andNMT8.0%oflutein,calculatedonthedriedbasis.
IDENTIFICATION
A.
Samplesolution:UsetheSamplesolutionfromthetestforContentofTotalCarotenoids.
Analysis:RecordtheUVVisspectrumfrom300600nm.
Acceptancecriteria:TheSamplesolutionshowsashoulderatabout428nm,anabsorption
maximumatabout450nm,andanothermaximumatabout478nm.
B.TheretentiontimeofthemajorpeakoftheSamplesolutioncorrespondstothatoftheStandard
solution,asobtainedinthetestforContentofZeaxanthin.
C.TheretentiontimeofthemajorpeakoftheSamplesolutioncorrespondstothatof3R,3R ,
carotene3,3diolfromtheStandardsolution,asobtainedinthetestforStereoisomeric
Composition.

http://www.usppf.com/pf/pub/index.html

1/6

6/30/2016

40(5)InProcessRevision:AztecMarigoldZeaxanthinExtract

COMPOSITION
CONTENTOFTOTALCAROTENOIDS
[NOTEUselowactinicglassware.]
Samplestocksolution:UsetheSamplestocksolutionfromthetestforContentofZeaxanthin.
Samplesolution:Transfer2.0mLoftheSamplestocksolutiontoa100mLvolumetricflask,
dilutewithethanoltovolume,andmixwell.
Instrumentalconditions
(SeeSpectrophotometryandLightScattering 851 .)
Analyticalwavelength:450nm
Cellpath:1cm
Blank:Ethanol
Analysis
Sample:Samplesolution
[NOTETheabsorbancereadingshouldbebetween0.2AUto1.0AU.Ifnot,readjustthe
dilutionofthesolution.]
Calculatethepercentageofthetotalcarotenoidsaszeaxanthin(C40H56O2):
Result=A/(CF)
A =absorbanceoftheSamplesolution
C =concentrationoftheSamplesolution(g/mL)
F =coefficientofextinction(E 1 %)ofzeaxanthininethanol(100mLg 1cm 1),2480

Acceptancecriteria:NLT36.0%oftotalcarotenoids(T)aszeaxanthin(C40H56O2)onthedried
basis
CONTENTOFZEAXANTHIN
[NOTEUselowactinicglassware.]
SolutionA:Methyltertbutylether
SolutionB:Water
SolutionC:Methanol
Diluent:Mixtureofhexane,ethanol,acetone,andtoluene(10:6:7:7)
Mobilephase:Gradientelution.SeeTable1.
Table1
Time
(min)

SolutionA
(%)

SolutionB
(%)

SolutionC
(%)

0.0

88

15

15

78

30

45

48

60

75

6.5

18.5

66

75

19

76

88

86

88

Standardstocksolution:Transfer20.0mgofUSPAztecMarigoldZeaxanthinExtractRSintoa
http://www.usppf.com/pf/pub/index.html

2/6

6/30/2016

40(5)InProcessRevision:AztecMarigoldZeaxanthinExtract

100mLvolumetricflask,add75mLofDiluenttotheflasktosuspendthesample,andsonicate
for5min.DilutewithDiluenttovolume,andmixwell.Allowtheinsolublestosettleforatleast
10min.Passthesupernatantthroughamembranefilterof0.45mporesize.
Standardsolution:Transfer0.5mLoftheStandardstocksolutiontoa8mLvial,evaporatedto
drynesswithanaidofastreamofnitrogen.Dissolvetheresiduein4.0mLofamixtureof
methyltertbutyletherandmethanol(5:95).
Samplestocksolution:Transfer20.0mgofExtracttoa100mLvolumetricflask,add75mLof
Diluenttotheflasktosuspendthesample,andsonicatefor5min.[CAUTIONElectrostatic
chargesmaycausethesampletosputterandsticktothesidesoftheflask.Carefullywash
downtheproductintotheflaskusingDiluent.]DilutewithDiluenttovolume,andmixwell.
Allowtheinsolublestosettleforatleast10min.Passthesupernatantthroughamembrane
filterof0.45mporesize.
Samplesolution:Transfer0.5mLoftheSamplestocksolutiontoa8mLvial,andevaporateto
drynesswiththeaidofastreamofnitrogen.Dissolvetheresidueina4.0mLmixtureof
methyltertbutyletherandmethanol(5:95).
Chromatographicsystem
(SeeChromatography 621 ,SystemSuitability.)
Mode:HPLC
Detector:450nm
Column:2.0mm15cm3mpackingL62
Flowrate:0.4mL/min
Injectionvolume:10L
Systemsuitability
Sample:Standardsolution
[NOTETheapproximaterelativeretentiontimesforluteinandzeaxanthinare0.87and1.0,
respectively.]
Suitabilityrequirements
Chromatographicsimilarity:ThechromatogramfromtheStandardsolutionissimilartothe
referencechromatogramprovidedwiththeUSPAztecMarigoldZeaxanthinExtractRSbeing
used.
Resolution:NLT1.0betweenzeaxanthinandlutein
Tailingfactor:NMT2.0forthezeaxanthinpeak
Relativestandarddeviation:NMT2.0%forthezeaxanthinpeak
Analysis
Sample:Samplesolution
Calculatethepercentageofalltranszeaxanthin(C40H56O2)intheportionofthesampletaken:
Result=(r U /r T )T
r U =peakresponseofalltranszeaxanthinfromtheSamplesolution
r T =sumoftheresponsesforallthepeaksfromtheSamplesolution
T

=percentageoftotalcarotenoidsasdeterminedinthetestforContentofTotal
Carotenoids

Acceptancecriteria:NLT30.0%ofalltranszeaxanthinonthedriedbasis
Changetoread:
http://www.usppf.com/pf/pub/index.html

3/6

6/30/2016

40(5)InProcessRevision:AztecMarigoldZeaxanthinExtract

LUTEINANDOTHERRELATEDCOMPOUNDS
[NOTEUselowactinicglassware.]
Mobilephase,Standardsolution,Samplesolution,Chromatographicsystem,andSystem
suitability:ProceedasdirectedinthetestforContentofZeaxanthin.
Analysis
Sample:Samplesolution
Calculatethepercentageofluteinintheportionofthesampletaken:
Result=(r U /r T )T
r U =peakresponseofluteinfromtheSamplesolution
r T =sumoftheresponsesforallthepeaksfromtheSamplesolution
T

=percentageoftotalcarotenoidsasdeterminedinthetestforContentofTotal
Carotenoids

Calculatethepercentageofotherrelatedcompoundsintheportionofthesampletaken:
Result=(r U /r T )100
r U =peakresponseofindividualrelatedcompoundspeaksfromtheSamplesolution
r T =sumoftheresponsesforallthepeaksfromtheSamplesolution

Acceptancecriteria
Lutein:NMT8.0%onthedriedbasis
Otherrelatedcompounds:NMT1.0%
2.0% 2S(USP38)
STEREOISOMERIC COMPOSITION
[NOTEUselowactinicglassware.]
Mobilephase:Gradientelution.SeeTable2.
Table2
Time
(min)

nHexane
(%)

2Propanol
(%)

0.0

95

50

95

55

50

50

63

50

50

65

95

75

95

Standardstocksolution:0.1mg/mLofUSPAztecMarigoldZeaxanthinExtractRSinmethylene
chloride.Sonicateifnecessarytodissolvethesample.
Standardsolution:Transfer1.0mLoftheStandardstocksolutionintoa15mLtesttube,and
evaporatetodrynesswiththeaidofastreamofnitrogen.Dissolvetheresidueina10.0mL
mixtureof2propanolandhexane(5:95).Passthroughamembranefilterof0.45mporesize.
Samplestocksolution:Weigh10mgofExtractintoa100mLvolumetricflask,add85mLof
methylenechloride,sonicate,andswirltodissolve.Dilutewithmethylenechloridetovolume.
Samplesolution:Transfer1.0mLoftheSamplestocksolutionintoa15mLtesttube,and
evaporatetodrynesswiththeaidofanitrogenstream.Dissolvetheresidueina10.0mL
mixtureof2propanolandhexane(5:95).Passthesolutionthroughamembranefilterof0.45
http://www.usppf.com/pf/pub/index.html

4/6

6/30/2016

40(5)InProcessRevision:AztecMarigoldZeaxanthinExtract

mporesize.
Chromatographicsystem
(SeeChromatography 621 ,SystemSuitability.)
Mode:HPLC
Detector:450nm
Column:4.6mm25cm5mpackingL51
Flowrate:0.8mL/min
Injectionvolume:20L
Systemsuitability
Sample:Standardsolution
[NOTETheapproximaterelativeretentiontimesfor(3R,3Smeso)zeaxanthin,(3R,3R)
zeaxanthin,and(3R,3R,6R)luteinare0.92,1.00,and1.12,respectively.]
Suitabilityrequirements
Resolution:Theresolutionbetweeneachpairofpeaksdueto(3R,3Smeso)zeaxanthin,
(3R,3R)zeaxanthin,and(3R,3R,6R)luteinisNLT1.0.
Chromatogramsimilarity:ThechromatogramfromtheStandardsolutionissimilartothe
referencechromatogramprovidedwiththeUSPAztecMarigoldZeaxanthinExtractRSbeing
used.
Analysis
Samples:StandardsolutionandSamplesolution
IdentifythepeaksoftherelevantanalytesinthechromatogramoftheSamplesolutionby
comparingwiththoseinthechromatogramoftheStandardsolutionobtainedfromtheSystem
suitability.
Calculatethepercentagesof(3R,3Smeso)zeaxanthinand(3R,3R)zeaxanthinintheportion
ofthesampletaken:
Result=(r U /r T )100
r U =peakresponseofthecorrespondinganalyte
r T =sumoftheresponsesfortwopeaks

Acceptancecriteria
(3R,3R)Zeaxanthin:NLT99.0%
(3R,3Smeso)Zeaxanthin:NMT1.0%
CONTAMINANTS
Deletethefollowing:
HEAVYMETALS,MethodII 231 :NMT20g/g

2S(USP38)

Addthefollowing:
ELEMENTALIMPURITIESP ROCEDURES 233
Acceptancecriteria
Arsenic:NMT0.5g/g
Cadmium:NMT1.0g/g
http://www.usppf.com/pf/pub/index.html

5/6

6/30/2016

40(5)InProcessRevision:AztecMarigoldZeaxanthinExtract

Lead:NMT5.0g/g
Mercury:NMT0.1g/g
2S(USP38)

Deletethefollowing:
RESIDUEONIGNITION 281 :NMT10.0%

2S(USP38)

SPECIFICTESTS
LOSSONDRYING 731
Analysis:Dryasampleundervacuumat105 for3h.
Acceptancecriteria:NMT25.0%
ADDITIONALREQUIREMENTS
P ACKAGINGANDSTORAGE:Preserveintight,lightresistantcontainers.
USPREFERENCESTANDARDS 11
USPAztecMarigoldZeaxanthinExtractRS
1S(USP37)

AuxiliaryInformationPleasecheckforyourquestionintheFAQsbeforecontactingUSP.
USP37NF32Supplement:No.1Page6525

http://www.usppf.com/pf/pub/index.html

6/6