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Home , Vitamin K2

Fat soluble detection in Food by LC/MS/MS

Stephen Lock, AB SCIEX, Europe.

INTRODUCTION Chromatography: Sampleswereseparatedbyreversed-

phaseHPLConasmallparticlesizeC18column(KinetexC18 fat soluble.rdb ( 1): "Linear" Regression ("No" weighting): y = 3.75e+004 x + 8.21e+004 (r = 0.9996) VITAMINs fat soluble.rdb (Vitamin D3 3): "Linear" Regression ("No" weighting): y = 3.85e+004 x + 1.5e+005 (r = 0.9986)

50x2.1mm,2.6µm,Phenomenex)at600µl/minusinga 1.9e7 2.0e7 Vitaminsareneededforhealthygrowthandtheseincludeagroupoffat 1.9e7 1.8e7 ShimadzuUFLCSystemovera7.5minutegradientfrom20% 1.8e7 solublevitaminsincludingA,D,EandK.VitaminAisimportant,itisa 1.7e7 1.7e7 1.6e7 sourceofmetaboliteswhichareimportantgrowthfactorsandalsoin methanolinwaterto100%methanol.Thecolumntemperature 1.6e7 1.5e7 Vitamin A 1.5e7 Vitamin D3 theformofretinaitisneededbytheeyeforbothlow-lightandcolour wasmaintainedat60ºCandaninjectionvolumeof50µLwas 1.4e7 1.4e7 1.3e7 vision. VitaminEisagainagroupofvitaminsofsimilarstructure. used. 1.3e7 1.2e7 1.2e7 1.1e7 ThereareeightdifferentformsofvitaminE,ofwhich γ-tocopherolis 1.1e7 1.0e7 commoninthedietandfoundinforexamplemargarinebutisnot 1.0e7 9.0e6 9.0e6 A r ea , c oun ts A r ea , c oun ts absorbedasefficientlybypeopleas α-,themostbiologically MassSpectrometry: AnalysiswasperformedonanAB 8.0e6 8.0e6 ® 7.0e6 activeformofvitaminEwhichisthesecondmostcommonformof SCIEXQTRAP 5500LC/MS/MSsystem. Thesource 7.0e6 6.0e6 6.0e6 vitaminE(foundinforexamplewheatgerm)andistheformnormally conditionswereastandardsetupofcurtaingas=40psi, 5.0e6 5.0e6 usedasanadditiveeitherinit’snativeformorastheacetateester. Nebulisercurrent=5kV(positivemode),gas1=30psiwith 4.0e6 4.0e6 3.0e6 3.0e6 VitaminEisanantioxidantthatstopstheproductionofreactiveoxygen thesourcetemperaturesetat400ºC. TheMRMconditions 2.0e6 2.0e6 usedareshowninTable1withtheresolutionkeptatunitfor 1.0e6 1.0e6 speciesformedwhenfatundergoesoxidationandhelpsreducecancer 0.0 0.0

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 risksfromsuchspecieswhichcancauseDNAdamage.VitaminKis bothQ1andQ3and ScheduledMRM™usedtoacquireallthe Concentration, ng/mL Concentration, ng/mL alsoagroupofstructurallysimilar,fat-solublevitaminsthatareneeded data. forthepost-translationalmodificationofcertainproteinsrequiredfor VITAMINs fat soluble.rdb (Vitamin K1 1): "Linear" Regression ("No" weighting): y = 3.47e+004 x + 8.29e+004 (r = 0.9999) VITAMINs fat soluble.rdb ( 1): "Linear" Regression ("No" weighting): y = 2.15e+004 x + 1.16e+004 (r = 0.9993) Q1 Q3 RT ID DP EP CE CXP bloodclottingandisinvolvedinmetabolicpathwaysofboneandother 1.8e7 1.10e7 1.7e7 1.05e7

1.00e7 tissue. ThetwonaturallyoccurringformsarevitaminK1andvitamin 1.6e7 9.50e6 [2] 269.2 93 4.1 Vitamin A 1 100 11 35 5 1.5e7 K2. 9.00e6 1.4e7 269.2 157.2 4.1 Vitamin A 2 100 11 41 15 8.50e6 1.3e7 Vitamin K1 8.00e6 ThefinalfatsolublevitaminisVitaminDandthiscanbeingestedas 1.2e7 7.50e6 Vitamin E 269.2 119.1 4.1 Vitamin A 3 100 11 31 5 7.00e6 1.1e7 (vitaminD3)orergocalciferol(vitaminD2)andthebody 6.50e6 1.0e7 canalsosynthesizeitfromcholesterolwhensunexposureissufficient. 451.4 187.1 4.7 Vitamin K1 1 65 12 37 15 6.00e6 9.0e6 5.50e6

8.0e6 5.00e6 VitaminD2canactuallybesynthesizedinadequateamountsbyall 451.4 128 4.7 Vitamin K1 2 65 12 116 9 A r ea , c oun ts A r ea , c oun ts 4.50e6 7.0e6 humansfromsunlight,howeverwhensunexposureislowandvitamin 4.00e6 451.4 199.2 4.7 Vitamin K1 3 65 12 45 5 6.0e6 3.50e6

Dinthedietisabsentdiseasescanoccurnamely,e.g.ricketsthe 5.0e6 3.00e6

458.4 136 4.7 Vitamin K1 IS 1 65 12 116 9 4.0e6 2.50e6 childhoodformofosteomalacia. Solikeothervitamins,vitaminDis 2.00e6 3.0e6 458.4 199.2 4.7 Vitamin K1 IS 2 65 12 45 5 1.50e6 addedtostaplefoodssuchasmilktoavoiddiseaseduetodeficiency. 2.0e6 1.00e6 ] 445.4 187.1 4.5 Vitamin K2 1 110 2 33 19 1.0e6 5.00e5 0.0 0.00 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 445.4 105 4.5 Vitamin K2 2 110 2 90 9 Concentration, ng/mL Concentration, ng/mL 445.4 80.9 4.5 Vitamin K2 3 110 2 71 11 Figure3. Theabovefiguresaretypicalcalibrationlinesthat 431.4 165.1 4.5 Vitamin E 1 120 9 40 15 canbeproducedinAPCImodewhenanalysingfatsoluble 431.4 137.1 4.5 Vitamin E 2 120 9 68 19 vitaminstandards. Allcalibrationlineswerelinearoverthe rangetested(1–250ppb)andhad‘r’valuesover0.99. 437.4 171.1 4.5 Vitamin E IS 1 120 9 40 15 Vitamin K2 (menaquinone) 437.4 143.1 4.5 Vitamin E IS 2 120 9 68 19 Vitamin K1 (Phylloquinone) 397.2 69.1 4.4 Vitamin D2 1 96 10 51 8 Figure4. Theopposite 397.2 91.1 4.4 Vitamin D2 2 96 10 83 8 XIC of +MRM (18 pairs): 269.200/92.956 amu Expected RT: 4.1 ID: vitamin A 1 from Sample 61 (meat + 10) of sample test.wiff (Hea... Max. 3.4e6 cps. figure shows an 400.2 69.1 4.4 Vitamin D2 IS 1 96 10 51 8 5.9e6 experiment where a 5.5e6 sampleofmincedmeat 400.2 91.1 4.4 Vitamin D2 IS 2 96 10 83 8 5.0e6 4.5e6 boughtfroma local 385.4 90.9 4.4 Vitamin D3 1 115 5 98 9 4.0e6 3.5e6 4.05 shop had been 385.4 367.4 4.4 Vitamin D3 2 115 5 21 17 3.0e6 extractedandanalysed 2.5e6

Vitamin (D3) I n t en s i y , c ps Vitamin D2 Meat sample Cholecalciferol 385.4 259.2 4.4 Vitamin D3 3 115 5 21 17 2.0e6 (bottompane).Asubset () 1.5e6 spiked at 10 ppm ofthesamesamplewas 388.4 90.9 4.4 Vitamin D3 IS 1 115 5 98 9 1.0e6 5.0e5 4.24 thenspikedat10ppm 388.4 259.2 4.4 Vitamin D3 IS 2 115 5 21 17 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 withamixtureofthefat Time, min XIC of +MRM (18 pairs): 269.200/92.956 amu Expected RT: 4.1 ID: vitamin A 1 from Sample 60 (meat) of sample test.wiff (Heated ... Max. 3.4e5 cps. soluble vitamins and 4.3e5 analysed. Theresults Table 2. The MRM conditions used to detect the Fat soluble 4.0e5 clearly shows the vitamins. 3.5e5 4.26

3.0e5 detectionofA,D,Eand

2.5e5 Kinthespikealthough Vitamin A (trans-) Vitamin E (α-Tocopherol) RESULTS 2.0e5 the meat was not

I n t en s i y , c ps 1.5e5 Blank Meat completely blank. 1.0e5 Figure 1. The chemical structures of the Fat soluble vitamins. sample 3.95 10ppmwasusedasthis XIC of +MRM (18 pairs): 431.400/165.100 amu Expect... Max. 2.4e4 cps. XIC of +MRM (18 pairs): 269.200/92.956 amu Expecte... Max. 5.1e5 cps. 5.0e4 3.89 4.06 isthelevelatwhich 0.0 4.79 4.04 2.4e4 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 some pet foods are 5.0e5 Time, min Traditionallyindividualmethodshavebeenusedtoscreenforeach spikedwithvitaminA 2.0e4 4.0e5 vitaminsoonemethodthatiscapabletoscreenforallfatsoluble andbelowthelevelof 1.5e4 4.54 3.0e5 vitaminswouldbebeneficial. Herewepresentsomenewdata 4.89 vitaminEadditives. acquiredbyLC/MS/MSwithascreeningmethodwhichcontains 1.0e4 Vitamin E 2.0e5 Vitamin A

I n t en s i y , c ps I n t en s i y , c ps CONCLUSIONS VitaminK1,K2,D2,D3,AandE.Detectionofthesevitaminshasbeen 5000.0 1.0e5 showntobeatthelowppblevelbyLC/MS/MSandreversephase 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 chromatographyusingpositivemodeatmosphericpressurechemical Time, min Time, min ThisworkhasclearlydemonstratedthatFatsolublevitaminscan ionisation.Themassspectrometrymethodsutilises Scheduled MRM™ XIC of +MRM (18 pairs): 385.400/90.900 amu Expecte... Max. 6.4e5 cps. XIC of +MRM (18 pairs): 397.221/69.100 amu Expecte... Max. 3.1e5 cps. bedetectedusingAPCIandLC/MS/MSanalysis. Althoughthe andasmallparticlesizeHPLCcolumnandsomefoodsampleswere responsesseenforeachvitaminvarywiththeweakestbeing 4.7e5 obtainedandthenextractedbyandanalysedbyLC/MS/MStoshow 8.0e5 vitaminE,theseresponsesactuallyalignwiththeRDAofthese 4.56 4.0e5 theapplicabilityofthismethodtoroutinesampleanalysis. 6.0e5 4.55 vitaminsinfoodsotheapproachofamultivitaminassayby 3.0e5 LC/MS/MSisvalidwithdetectionlevelsofsomevitaminsbelow1 4.0e5 Vitamin D3 2.0e5 Vitamin D2 ppb. Inthisoriginalworkamethodtodevelopthenativevitamins I n t en s i y , c ps I n t en s i y , c ps MATERIALS AND METHODS 2.0e5 1.0e5 hasbeendevelopedandthedetectionlimitinfoodwilldepend 0.0 0.0 mainlyontheextractiontechniquesused. Forlowlevelvitamins 1.0 2.0 3.0 4.0 5.0 6.0 7.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Samplepreparation: Sample(1g)wasmixedwithmethanol(1mL) Time, min Time, min e.g.vitaminD,asolidphaseextractionmayberequired(whichhas containingpyrogallol(3%,w/v)and50%aqueousmethanolwasadded XIC of +MRM (18 pairs): 451.400/187.100 amu Expect... Max. 1.2e6 cps. XIC of +MRM (18 pairs): 445.400/187.100 amu Expect... Max. 5.4e5 cps. beendevelopedbutnotdiscussedinthispresentation)whilefor 5.15 4.73 highlevelvitaminssuchasAandEthesimplesaponificationand (3mL)togetherwithinternalstandardsolution(concentration1ppm, 1.21e6 5.0e5 liquid/liquidextractionissufficient.Infutureworktheesterforms 100µL).Thesamplewassonicated(10minutes)andshakenorroller 1.00e6 mixforafurther20minutes. KOHwasthenaddedandthesample 4.0e5 ofvitaminAandEareplannedtobeaddedtothismethodasthese Vitamin K1 Vitamin K2 2 vortexmixed.Saponification,tobreakupthefatsandreleasethe 3.0e5 areoftentheformsatwhichthevitaminsareusedasadditives . 5.00e5 vitamins,wasthenperformedat40ºCfor30minduringwhichthe 2.0e5 I n t en s i y , c ps I n t en s i y , c ps sampleswerebrieflyvortexmixed.Thesampleswerethencooledand 1.0e5 REFERENCES acidifiedwithasolutionofHCL 0.00 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Time, min Time, min 1. http://www.food.gov.uk/multimedia/pdfs/labellingfoodsupplement Hexane(15mL)wasaddedandthesamplesvigorouslyshakenand s.pdf. vortex-mixedfor20sthreetimeswiththetubeskeptcoolbetween 2.Directive 2002/46/EC of the European Parliament. mixing.Attheendofthehexaneextractionthetubeswerecentrifuged Figure2. The20ppbsolventstandardaboveisamixtureof (3000rpm,5minutes).Thetoplayer(3ml)wastakenandevaporate thefatsolublevitamins. Inthisfigureyoucanseethatthe todryness.Thesamplewasreconstitutedwithacetonitrile(0.5ml)and responseforeachvitaminvarieswithVitaminEtheweakest TRADEMARKS/LICENSING vortexed(30s),sonicated(5mins)andvortexed(30s)andthenthis with20ppbit’slimitofdetection(LOD).HowevervitaminE’s extractwasdilutedwith50%aqueousacetonitrile(2ml).Thissample recommendeddailydoes(RDA)isa1000xhigherat12mg For Research Use Only. Not for use in diagnostic procedures. preparationwasdesignedtodetectvitaminsatlevelsfoundinfoods comparedtothemostactivevitamin,vitaminD,whoseRDAis The trademarks mentioned herein are the property of AB SciexPte. 1 (0.1–50ppm)andforlowerlevelsaSPEextractionhasalsobeen 5µgwhilevitaminARDAis800µgandvitaminKis75µg . Ltd. or their respective owners. developedtodetectthesevitaminsbutthisisnotdiscussedinthis TheseRDAaresetinEuropebytheEuropeanCommission AB SCIEX™ is being used under license. presentation. (CommissionDirective2008/100/EC).ThereforethevitaminE levelwillbeovera1000timeshigherthanvitaminDinfoodso © 2013 AB SCIEX. it’sLODcanbesetalothigher. InthismethodvitaminD’s LOD,inboththeD2andD3form,isbelowappb.