analysis of water soluble vitamins by prominence system
TRANSCRIPT
Analysis of Water-Soluble Vitamins Using the Prominence System
Contents1. Introduction………………………………………………………………………………………… 1
2. SimultaneousAnalysisofVitaminBGroup… …………………………………………………… 22.1 TargetComponents… ………………………………………………………………………… 22.2 UnitConfiguration……………………………………………………………………………… 22.3 OptimizingtheAnalyticalConditions………………………………………………………… 2
2.3.1 SettingtheSeparationConditions……………………………………………………… 22.3.2 SettingtheDetectionWavelength……………………………………………………… 32.3.3 StandardAnalyticalConditions………………………………………………………… 4
2.4 SamplePreparation… ………………………………………………………………………… 52.5 AnalysisExamples… ………………………………………………………………………… 6
3. AnalysisofOtherWater-SolubleVitamins………………………………………………………… 83.1 SettingtheDetectionWavelength……………………………………………………………… 83.2 AnalysisofCyanocobalamin…………………………………………………………………… 93.3 AnalysisofAscorbicAcid……………………………………………………………………… 103.4 AnalysisofCarnitine…………………………………………………………………………… 113.5 AnalysisofHesperidin………………………………………………………………………… 12
4. Conclusions………………………………………………………………………………………… 13
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1. Introduction
Vitaminsareessentialnutrientsand,recently,theyhavebeensupplementedwithmedicationsorsupplements.
Theanalysismethodforquantitationofvitaminsisshiftingfromabiologicalwaytoachemicalway,andhigh-
performanceliquidchromatography(HPLC)iswidelyusedastheanalysismethod.
Thispaperreportsontheanalysismethodsforwater-solublevitaminsinmedicationsorvitaminsupplements
(Figure1)usingtheProminenceHPLCSystem.
Fig. 1StructuresofWater-SolubleVitamins
Riboflavin (B2)
Hesperidin (P)
Carnitene (BT)Ascorbic Acid (C)
Folic Acid (M)
Nicotinamide (B3, B5, PP)Nicotinic Acid (B3, B5)
Pantothenic Acid (B3, B5)
Cynanocobalamine (B12)
Biotin (H)
Thiamin (B1)
Riboflavin phosphate (B2)
Pyridoxine (B6)
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2. Simultaneous Analysis of Vitamin B Group
2.1 Target Components
ThevitaminBgroupincludesmanycomponents.Weinvestigatedanewmethodforsimultaneousanalysis
ofninewater-solublevitamins(thiamin(vitaminB1),riboflavinandriboflavinphosphate(bothvitaminB2),
pyridoxine(vitaminB6),nicotinicacid(niacin),nicotinamide(niacinamide),pantothenicacid(vitaminB5),
biotin(vitaminB7orH),andfolicacid(vitaminB9orM))andcaffeine.
2.2 Unit Configuration
TheProminencesystemwasusedfor thisstudy:LC-20ADSolventDeliveryUnit,CTO-20ACColumn
Oven,SPD-20AVUV-VISDetectorandSPD-M20APhotodiodeArrayDetector,SIL-20ACAutosampler,
andLCsolutionWorkstation.
2.3 Optimizing the Analytical Conditions
2.3.1 Setting the Separation Conditions
Toinvestigatesimultaneousanalysisof thisvitaminBgroupusingreverse-phasechromatography,
whichisthemostwidelyusedwithHPLCanalysis, theseparationconditionsmustbesettoachieve
goodseparationwithanappropriateratioofretentionbetweenthiaminandpyridoxine,whichhave
weakretention,andriboflavin,whichhasstrongretention.Thiamin isastronglybasicsubstance
containingquaternarynitrogeninitsmoleculesandcarriesapositivechargeinthemobilephase.It
oftenpassesthroughthereverse-phasecolumn,asnohydrophobicinteractionoccurswithasolidphase.
Thisproblemcanbesolvedbyaddinganion-pairreagentwithnegativechargeandahydrophobic
function to themobilephase.Thisreagent ishydrophobicallyretainedby thesolidphasewhere it
causesanionicinteractionwiththetargetcomponents,allowingreverse-phaseion-pairchromatography
tobeused.Alkylsulfonatesareoftenusedasion-pairreagentsforstronglybasicsubstances,suchas
sodiumoctanesulfonateorsodiumpentasulfonate.Generally, theretentionof thetargetcomponents
increasesasthelengthofthealkyl-chainintheion-pairreagentgetslongerandastheion-pairreagent
concentrationrises(uptoacertainconcentration).Conversely,asriboflavinhasstrongretention,an
organicsolvent(acetonitrileormethanol)mustbeaddedtothemobilephasetoaccelerateitselution.
Asdescribedabove, in thecaseof thereverse-phasechromatographyfor thisvitaminBgroup, the
ion-pairreagentmustbeaddedtoselectivelystrengthentheretentionofthiaminandpyridoxine,and
theratiooforganicsolventinthemobilephasemustbecontrolledtoreducetheanalysistime,while
ensuringtheseparationofallcomponents.
Forthisanalysis,themobilephaseconditionswereoptimizedusingaShim-packVP-ODS(4.6mmID
x150mmlong)reverse-phasechromatographycolumnaccordingtotheprocedurebelow.
1)Tostrengthentheretentionofpantothenicacid,riboflavinphosphate,andfolicacid,thepHofmobile
phasewassettotheacidicsideandtheorganicsolventconcentrationwasreduced.Inaddition,the
saltconcentrationwasincreasedinconsiderationofthesalting-outeffect.
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2)Alkylsufonateswereaddedasanion-pairreagenttothemobilephasetoretainbasicsubstancessuch
asthiamin,pyridoxine,andbiotin.Weinvestigatedtheretentionbehaviorofbasicsubstancesusing
severaltypesofalkylsulfonatewithdifferentchainlengthsforseveralcombinationsoftheorganic
solventconcentration(setin1)aboveandalkylsulfonateconcentration.
Asaresultof this investigation,weadoptedamobilephasecomprising100mmol/Lphosphoric
acid(sodium)buffersolution(pH2.1)�containing0.8mmol/Lsodiumoctanesulfonatemixedwith
acetonitrile(mixingratio19/2).
2.3.2 Setting the Detection Wavelength
Figure2showstheUVabsorptionspectraforthevitaminBgroupandcaffeineusingthemobilephase
setinsection2.3.1.Table1showsthewavelengthofmaximumabsorbanceforeachcomponent.The
pantothenicacidwasdetectedatashortwavelengthof210nm,whileothercomponentshavingenough
absorptionnear250to300nmweredetectedat270nmwhereimpuritycompornentshadlessofan
adverseeffect.SimultaneousdetectionispossibleusingtheSPD-M20Aphotodiodearraydetectoror
usingtheSPD-20A/20AVUV-VISdetector’stwo-wavelengthsimultaneousmeasurementfunction.
Fig. 2UVAbsorptionSpectraoftheVitaminBGroupandCaffeine
Table 1WavelengthofMaximumAbsorbancefortheVitaminBGroup
Component WavelengthofMaximumAbsorbance(nm)Thiamin 192,247Riboflavin 190,223,267,373RiboflavinPhosphate 190,223,267,373Pyridoxine 190,290NicotinicAcid 192,210,260Nicotinamide 192,210,260PantothenicAcid 192Biotin 192FolicAcid 195,285
�Howtoprepare100mmol/Lphosphoricacid(sodium)buffersolution(pH2.1)•Sodiumdihydrogenphosphatedehydrate(MW=156.01) 50mmol(7.8g)•Phosphoricacid(85%,14.7mol/L) 50mmol(3.4mL)•Dissolvetheaboveinwaterandmakeupto1000mL.
Pantothenic AcidBiotin
Pyridoxine
Caffeine
Riboflavin Phosphate
Nicotinic Acid
Folic Acid
Riboflavin
Thiamin
Nicotinamide
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2.3.3 Standard Analytical Conditions
ThestandardanalyticalconditionsshowninTable2weresetbasedontheinvestigationresults.
Table 2AnalyticalConditions
Column : Shim-packVP-ODS(150mmL.x4.6mmi.d.)MobilePhase : A)100mmol/L(Sodium)phosphatebuffer(pH2.1)
containing0.8mmol/Lsodium1-octanesulfonateB)Acetonitrile
A/B=19/2(v/v)FlowRate : 1.2mL/minColumnTemp. : 40°CDetection : 210nm,270nm
Figure3showstheanalysisresultsofastandardmixtureofthevitaminBgroupandcaffeine.
Fig. 3ChromatogramsofaStandardMixtureoftheVitaminBGroupandCaffeine
Peaks 1. Niacin 2. Nicotinamide 3. Ca Pantothenate 4. Pyridoxine 5. Riboflavin Phoshate 6. Thiamin 7. Caffeine 8. Folic Acid 9. Biotin10. Riboflavin
(20mg/L each, 10μL inj.)
1 2
4
5
7
6
8
10
270nm
1
2 4
35
7
6 8 10
9
210nm
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2.4 Sample Preparation
Table3showsthesolubilityandstabilityofthevitaminBgroupandcaffeine.Consideringthatthiaminand
pyridoxinehavestrongadsorptiontotheglasssurfaceandsamplematrix,anacidsolutioniseffectivefor
samplepreparation.However,riboflavin,riboflavinphosphate,biotin,andfolicacidhavepoorsolubility
belowneutralpH.Therefore, theywerefirstdissolvedinanaqueoussodiumhydroxidesolutionandthen
dilutedwithanacidicsolution.Thesamplemustbepreparedinthedarkasmanycomponentsareunstableto
light.
Table 3SolubilityandStabilityofVitaminBGroupandCaffeine
CompoundNameSolubility Stability
Acidic WeaklyBasic Acidic WeaklyBasic LightThiamin ○ ― ○ × ×Riboflavin × ○ ○ × ×RiboflavinPhosphate ― ○ ○ × ×Pyridoxine ○ ― ○ ― ×NicotinicAcid ○ ― ○ ○ ○Nicotinamide ○ ― ○ ○ ○PantothenicAcid ○ ― ○ ○ ○Biotin ― ○ ○ ― ○FolicAcid × ○ ○ × ×Caffeine ○ ― ○ ○ ○
(○:good×:bad―:unknown)
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2.5 Analysis Examples
Figure4showsanexampleoftheanalysisofacommercialvitamintablet(dietarysupplement).100mgof
thepulverizedtabletwasaddedto20mL100mmol/Lsodiumhydroxidesolutionandsubjectedtoultrasonic
extractionfor10minutes.Aftercentrifuging,1mLsupernatantwasmadeupto50mLwithmobilephase,
andfilteredthrougha0.45μmmembranefilter.10μLofthissamplewasinjectedforanalysis.
Fig. 4ChromatogramsofaVitaminTablet
Peaks 1. Nicotinamide 2. Ca Pantothenate 3. Pyridoxine 4. Thiamin 5. Riboflavin
2
4
5
1
3 210nm
270nm
4
5
1
3
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Figure5showsanexampleoftheanalysisofacommercialdrink(nonmedicinalproduct).1mLofthesample
wasmadeupto10mLwithmobilephase,andfilteredthrougha0.45μmmembranefilter.10μLofthis
samplewasinjectedforanalysis.
Fig. 5ChromatogramsofaVitaminDrink
Peaks 1. Nicotinamide 2. Pyridoxine 3. Riboflavin Phoshate 4. Thiamin 5. Caffeine
270nm
210nm
2
4
1
3
5
2
4
1
3
5
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3. Analysis of Other Water-Soluble Vitamins
Thissectionintroducessomeexamplesoftheanalysisofothermajorwater-solublevitamins:cyanocobalamin
(vitaminB12),ascorbicacid(vitaminC),carnitine(vitaminBT),andhesperidin(vitaminP).
3.1 Setting the Detection Wavelength
Figure6showstheUVabsorbancespectrumforeachcomponent.Table4showsthewavelengthofmaximum
absorbanceforeachcomponent.
Fig. 6UVAbsorptionSpectraofWater-SolubleVitamins
Table 4WavelengthofMaximumAbsorbanceforWater-SolubleVitamins
Component WavelengthofMaximumAbsorbance(nm)Cyanocobalamin 190,278,362,550AscorbicAcid 243Carnitine 190,204Hesperidin 199,283
Cyanocobalamin
Hesperidin
Ascorbic Acid
Carnitine
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3.2 Analysis of Cyanocobalamin
Ascyanocobalaminisstronglyretainedinareverse-phasechromatographycolumn,acetonitrilewasadded
tothemobilephasetoreducetheelutiontime.Asthecontentofcyanocobalamininvitamintabletsisonly
1/1000to1/3000of theothercomponent, itwasnecessarytoset thedetectionwavelengthto550nmto
inhibittheeffectsoftheinterferingcomponents,andtoincreasetheabsoluteamountofcyanocobalaminfor
analysis.Figure7showsthechromatogramofacyanocobalaminstandardsolution(10mg/L,10μLinjected
volume)undertheanalyticalconditionsshowninTable5.Figure8showsthechromatogramofacommercial
vitamintablet(supplement).9.6gofthepulverizedtabletwereaddedto10mL1mmol/Lsodiumhydroxide
solutionandsubjectedtoultrasonicextractionfor10minutes.Thiswasmadeupto100mLwithmobile
phaseA,subjectedtoultrasonicextractionfor10minutes,andfilteredthrougha0.45μmmembranefilter.
10μLofthissamplewasinjectedforanalysis.
Table 5AnalyticalConditions
Column : Shim-packVP-ODS(150mmL.x4.6mmi.d.)MobilePhase : A)100mmol/L(Sodium)phosphatebuffer(pH2.1)
B)AcetonitrileA/B=8/1(v/v)
FlowRate : 1.2mL/minColumnTemp. : 40°CDetection : 550nm
Fig. 7ChromatogramofCyanocobalamin Fig. 8ChromatogramofVitaminTablet
Peaks 1. Cyanocobalamimn
1
1
Peaks 1. Cyanocobalamimn
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3.3 Analysis of Ascorbic Acid
Ascorbicacidcanbeanalyzedbyreversed-phasechromatographywithtetrabutylammoniumaddedasan
ion-pairreagent.Generally,however,ascorbicacidisanalyzedbynormal-phasechromatographyusingan
NH2column,whichprovidesgoodseparationfromothervitamins.Figure9showsthechromatogramofan
ascorbicacidstandardsolution(100mg/L,10μLinjectedvolume)undertheanalyticalconditionsshownin
Table6.Figure10showsthechromatogramforacommercialsoftdrink.Thesamplewasdiluted100times
in1%(w/v)metaphosphoricacidandfilteredthrougha0.45μmmembranefilter.10μLofthissamplewas
injectedforanalysis.
Table 6AnalyticalConditions
Column : NH2P-504E(250mmL.×4.6mmi.d.)MobilePhase : A)100mmol/L(Triethanolamine)phosphatebuffer(pH2.2)�
B)AcetonitrileA/B=1/4(v/v)
FlowRate : 1.0mL/minColumnTemp. : 40°CDetection : 240nm
* phosphoricacid6.8mL+triethanolamine7.46g+water→total1000mL
Fig. 9ChromatogramofAscorbicAcid Fig. 10ChromatogramofSoftDrink
Peaks 1. Ascorbic Acid
Peaks 1. Ascorbic Acid1
1
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3.4 Analysis of Carnitine
Ascarnitine isweakly retained ina reverse-phasechromatographycolumnand tailingoccurs readily,
sodiumperchloratewasaddedtothemobilephaseunderanacidicconditiontoincreasetheretentioneffect
andrestricttailing.Figure11showsthechromatogramofacarnitinestandardsolution(1000mg/L,10μL
injectedvolume)undertheanalyticalconditionsshowninTable7.
Table 7AnalyticalConditions
Column : Shim-packVP-ODS(150mmL.x4.6mmi.d.)MobilePhase : A)10mmol/L(Sodium)phosphatebuffer(pH2.6)
containing0.2mol/LsodiumperchlorateFlowRate : 1.0mL/minColumnTemp. : 40°CDetection : 210nm
Fig. 11ChromatogramofCarnitine
1 Peaks 1. Carnitine
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3.5 Analysis of Hesperidin
Ashesperidin is strongly retained ina reverse-phasechromatographycolumn, likecyanocobalamin,
acetonitrilewasadded to themobilephase for theanalysis.Figure12 shows thechromatogramofa
hesperidinstandardsolution(100mg/L,10μLinjectedvolume)undertheanalyticalconditionsshownin
Table8.
Table 8AnalyticalConditions
Column : Shim-packVP-ODS(150mmL.x4.6mmi.d.)MobilePhase : A)100mmol/L(Sodium)phosphatebuffer(pH2.1)
B)AcetonitrileA/B=3/1(v/v)
FlowRate : 1.5mL/minColumnTemp. : 40°CDetection : 265nm
Fig. 12ChromatogramofHesperidin
1 Peaks 1. Hesperidin
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4. Conclusions
Analysisexamplesof thevitaminBgroupandotherwater-solublevitaminsusing theProminencesystem
wereintroduced.Thesemedicationsorvitaminsupplementscanbeanalyzedaftersimplepretreatment,asthey
containrelativelylowlevelsofimpuritycomponentsandhighlevelsofthetargetcomponents.However,some
skillisrequiredwiththedetectionmethodandpretreatmentmethodfortheanalysisofnaturalsamples.Insome
cases,post-columnderivatizationisusedtoenhancesensitivityandselectivityfortheanalysisofwater-soluble
vitamins.Theseanalyseswerepreviouslyreported inShimadzuApplicationNews,andwe intend tokeep
studyingandintroducingtheminApplicationReportsinthefuture.