Analysis of Water Soluble Vitamins by Prominence System

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.



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.


Column : NH2P-504E(250mmL.×4.6mmi.d.)MobilePhase : A)100mmol/L(Triethanolamine)phosphatebuffer(pH2.2)�



* 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.



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.





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.

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Analysis of Water Soluble Vitamins by Prominence System