Plant-Based Beverages as Good Sources of Free and Glycosidic Plant Sterols

18 Pages·2017·1.1 MB·English

nutrients
Article
Plant-Based Beverages as Good Sources of Free
and Glycosidic Plant Sterols
AnneleenIDecloedt1,2,*,AnitaVanLandschoot1,3,HellenWatson1,DanaVanderputten3
andLynnVanhaecke2
1 FacultyofBioscienceEngineering,LaboratoryofBiochemistryandBrewing,GhentUniversity,
ValentinVaerwyckweg1,B-9000Ghent,Belgium;[email protected](A.V.L.);
[email protected](H.W.)
2 FacultyofVeterinaryMedicine,DepartmentofVeterinaryPublicHealthandFoodSafety,Laboratoryof
ChemicalAnalysis,GhentUniversity,133Salisburylaan,B-9820Merelbeke,Belgium;
[email protected]
3 FacultyofScienceandTechnology,DepartmentofBiosciencesandFoodSciences,UniversityCollegeGhent,
ValentinVaerwyckweg1,B-9000Ghent,Belgium;[email protected]
* Correspondence:[email protected];Tel.:[phone]
Received:25October2017;Accepted:18December2017;Published:29December2017
Abstract: To address the ever-growing group of health-conscious consumers, more and more
nutritionalandhealthclaimsarebeingusedonfoodproducts. Nevertheless,onlyveryfewfood
constituents, including plant sterols, have been appointed an approved health claim (European
Commission and Food and Drugs Administration). Plant sterols are part of those limited lists
ofapprovedcompoundsfortheircholesterol-loweringpropertiesbuthavebeenpraisedfortheir
anti-inflammatoryandanti-carcinogenicpropertiesaswell. Despitethisindisputablereputation,
directquantitativedataisstilllackingfornaturallypresent(conjugated)plantsterolsinbeverages.
ThisstudyaimedtofillthisgapbyapplyingavalidatedextractionandUPLC-MS/MSdetection
methodtoadiverserangeofeverydayplant-basedbeverages. β-sitosterol-β-D-glucoside(BSSG)
showedtobebyfarthemostabundantsterolinallbeveragesstudied,withconcentrationsupto60–90
mgper100mLinplant-basedmilkalternativesandfreshfruitjuices. Ergosterol(provitaminD )
2
couldbefoundinbeers(0.8–6.1µgper100mL,fromtheyeast)andoccasionallyinjuices(17–29µgper
100mL).Overall,theresultsdemonstratedthattheconcentrationsofwater-solublesterolconjugates
havebeenunderestimatedsignificantlyandthatspecificplant-basedbeveragescanbegood,low-fat
sourcesoftheseplantsterols.
Keywords: (conjugated) plant sterols; beverages; cholesterol-lowering; ergosterol; anti-aging;
coronaryheartdisease;healthclaims;anti-inflammatory;anti-carcinogenic
1. Introduction
Overthelastdecade,thebeverageindustryhastakenaseriousjumpintothefuturebyintroducing
abroadrangeofnew,convenient,natural,andfunctionalbeverages. Thesebeverages,oftencalled
healthdrinks,include(iced)teasandjuicesbutalsoshakesand“superdrinks”suchaspomegranate
juiceorAloeveraextract–baseddrinks. Formostoftheseproducts,arangeofdifferentnutritionand
healthclaimsarebeingformulatedonthelabelandinadvertisements. Theseclaimsstimulatethe
consumer to purchase these particular beverages in order to increase their personal health status.
Popularhealthclaimsare“freefrom”claimssuchasgluten-freeorlactose-freebutalsovegan,organic,
“helpstopreventcoronaryheartdisease”and“lowerscholesterol”claimsarebeingusedquiteoften.
Tostreamlinetheseclaims,FDA(FoodandDrugAdministration)andEC(EuropeanCommission)
ruleshavebeenadoptedontheuseofnutritionandhealthclaimsonfoods. Upuntilnow,onlyvery
Nutrients2018,10,21;doi:10.3390/nu10010021 www.mdpi.com/journal/nutrients
Nutrients2018,10,21 2of18
Nutrients 2017, 10, 21   2 of 18 
fewcompoundshavebeenappointedanapprovedhealthclaimbybothorganizations.Plantsterolsare
Up until now, only very few compounds have been appointed an approved health claim by both 
partofthatlimitedlistofapprovedcompoundsfortheircholesterol-loweringproperties(FDAHealth
organizations.  Plant  sterols  are  part  of  that  limited  list  of  approved  compounds  for  their 
Claim;PhytosterolsandRiskofCoronaryHeartDisease)(EFSA,Article14(1)(a)“Reductionofdisease
cholesterol‐lowering properties (FDA Health Claim; Phytosterols and Risk of Coronary Heart 
risk” of the Regulation on nutrition and health claims 1924/2006). Less well-known, but perhaps
Disease) (EFSA, Article 14 (1)(a) “Reduction of disease risk” of the Regulation on nutrition and 
evenmorepromising,arearangeofothersuggestedhealthbenefitsrelatedtotheconsumptionof
health claims 1924/2006). Less well‐known, but perhaps even more promising, are a range of other 
plantsterolssuchasanti-carcinogenic,anti-inflammatory,andanti-oxidativeeffects[1–6]. Despitethis
suggested health benefits related to the consumption of plant sterols such as anti‐carcinogenic, 
generalinterest,quantitativedataontheconcentrationofthesesterolsinday-to-dayconsumption
anti‐inflammatory, and anti‐oxidative effects [1–6]. Despite this general interest, quantitative data on 
goothdes aconndceensptreactiiaolnl yobf etvheesrea gsetesraorles einx tdreamy‐etoly‐dsacya rccoen.sumption goods and especially beverages are 
exPtrleamntelsyt escroarlsc,e.o r phytosterols, are one of the main constituents of plant membranes, playing
animpoPrltaanntt srtoelreolisn, ocer lplhmyetomsbterraonlse, satraeb oilnitey oafn tdhea mssaiignn caolntrsatintusdenutcse orsf p[7la].nEt rmgeomstberroanle(ps,r opvlaiytainmgi nanD 2),
onitmhepoortthaenrt hroanled i,nt ackelels muepmabsriamneil astrarboillietyin anpdro atso zsoiganaanl dtrafunsndguic(eer.gs .[,7y].e Easrtg)oasntedroils (approrvoivtaitmamini nD2f)o, rm
ofvointa tmhei notDhero hraenrdg,o tcaaklecsif uerpo al. sEimxpiloasru rroeleo ifne prgrootsotezrooal atnodu flutrnagvii o(el.egt.,( UyeVa)stl)i gahntdc iasu as pesroavpithamotionc hfoermmi cal
2
reaocft iovnitathmaitn pDro2 dourc eesrgvoitcaamlciifnerDol2. . ETxhpoissuhraep  poef nesrgnoasttuerraolll ytot oualtrcaevritoalient e(xUtVen) t,liagnhdt  cqauuistees ofat en,
mupshhorotoocmhesmariceailr rraeadcitaitoend tahfatte rphroadrvuecsets tvoitianmcriena sDe2.t hTehiirsV hiatapmpeinnsD nactuornatlelny tt[o8 ]a. Fceurntagiina erxetaenlsto, agnrodw  n
2
indquusittrei aolfltyens,o mtuhsahtreorogmoss taerreo lircraadniabteede xatfrtearc theadrvaensdt toco innvcreeratseed thtoeiVr iVtaitmamininD D2 fcoornsteanlet [a8s]. aFudniegtia ry
2
supaprele amlseon gtroawndn ifnodoudstardiadlliyt isvoe t[h9a]t. erCghoestmeriocla cllayn, beerg eoxsttrearcoteld( panrodv ciotanmveinrteDd t)o aVnidtamplinan Dt2s ftoerr osalslea re
2
verays aal idkeietaanrdy ssuimppillaermteontth aenirdh fuomoda nadadnidtivaen [i9m].a Clchoeumnictearllpya, retr,gcohsotelerostl e(rporlo(vFitiagmurien 1D).2)T ahnedy pcloanntta in
sterols are very alike and similar to their human and animal counterpart, cholesterol (Figure 1). They 
astereo-specificorientedmethylorethylsubstitutionattheC24positionofthesterolsidechainand,
contain a stereo‐specific oriented methyl or ethyl substitution at the C24 position of the sterol side 
inthecaseofstigmasterol,ergosterol,andbrassicasterol,anadditionaldoubleboundbetweenC22
chain and, in the case of stigmasterol, ergosterol, and brassicasterol, an additional double bound 
andC23[4,10](Table1). Uponhumanconsumption,thesestructuralandfunctionalresemblances
between C22 and C23 [4,10] (Table 1). Upon human consumption, these structural and functional 
allowplantsterolsandergosteroltointerferewithcholesterolabsorptionintheintestinaltractthrough
resemblances allow plant sterols and ergosterol to interfere with cholesterol absorption in the 
displacementofcholesterolfromthemicellesand/orcompetitionwithcholesterolbindingproteins.
intestinal tract through displacement of cholesterol from the micelles and/or competition with 
Ascahorleessutelrt,oll obwin-ddienngs iptyroltiepinosp. roAtse ian r(eLsDuLlt,) lcohwo‐ldesetnesriotyl lleipvoeplsrowteiilnl d(LecDrLea) sceh,olloewsteerroinl gletvheels rwiskill for
cordoencarreyashee, alorwtfeariilnugr eth[1e 1r,i1s2k] f.or coronary heart failure [11,12]. 

FigFuirgeu1re. C1.h Cehmemicaiclaslt srturcutcuturereo offt htheem maaiinn ffrreeee aanndd ccoonnjujuggaatetedd pplalnatn sttsetreorlos l(sca(cmapmepsteesrtoelr, oslt,igsmtigamstearsotle, rol,
brabsrsaicssaisctaesrtoelr,oβl, -βsi‐tsoitsotsetreorlol( B(BSSSS),),a annddβ β-‐ssiittoosstteerrooll‐-ββ‐-DD‐g-glulucocosisdied e(B(SBSSGS)G),) )c,hcohleoslteesrtoelr o(aln(iamnaiml satelrsotel)r, ol),
andanedrg eorsgtoesrtoelro(flu (fnugnagl/aly/yeeaasstts stteerrooll,, pprroovviittaammiinnee DD2)). .
2
Nutrients2018,10,21 3of18
Table1.Chemicalsimilaritiesandstructuraldifferencesbetweencholesterol,ergosterol(provitamin
D )andplantsterols.Listedcharacteristicsincludemeanmass,molecularformula,numberofdouble
2
bounds,positionofdoublebounds,andalkylgrouppresentatC24(none,methyl,orethyl).
StructuralCharacteristics
SterolName MeanMass(Da) MolecularFormula PositionDouble
DoubleBounds AlkylGroupatC24
Bounds
Cholesterol 386.654 C27H46O 1 C5–C6 /
C5–C6
Ergosterol
(ProvitaminD2) 396.648 C28H44O 3 CC227––CC283 Methyl
β-sitosterol-β-D-glucoside
(BSSG) 576.847 C35H60O6 1 C5–C6 Ethyl
β-sitosterol
(BSS) 414.707 C29H50O 1 C5–C6 Ethyl
C5–C6
Brassicasterol 398.675 C28H46O 2 C22–C23 Methyl
C5–C6
Stigmasterol 412.691 C29H48O 2 C22–C23 Ethyl
Campesterol 400.691 C28H48O 1 C5–C6 Methyl
TheEuropeanFoodSafetyAuthority(EFSA)andFDAconcludedthat,relativetoaplacebo,blood
LDLcholesterollevelscanbereducedby7to12.5%ifapersonconsumes1.5to3gramsofplantsterols
andstanols(expressedasfreesterols)aday(EFSAclaim,article14(1)(a))[13,14].Arecentmeta-analysis
by Ras et al. (2014) (124 studies, 202 stratas) extended these findings; they found that plant sterol
intakesof0.6to3.3gperdaygraduallyreduceLDL-cholesterolconcentrationsby,onaverage,6to
12%[15]. Thecholesterol-loweringeffectisusuallyestablishedwithintwotothreeweeksafterdiet
changeandcouldbesustainedformonths[16]. Nosignificantalterationsinhigh-density-lipoprotein
(HDL)-cholesterol(the“good”cholesterol),ortriglyceridesingeneral,werereported. Effectivenessof
thisapproachhasbeenpositivelytestedinhypercholesteraemicpatientsaswellasinindividualswith
normalcholesterollevels[17].
FDAandECrulesonnovelfoodandnovelfoodingredients(inforcesince1997)requireallnew
ingredientstogothroughanapplicant-specificauthorizationprocedurethatinvolvesarigoroussafety
assessmentbeforetheycanbeplacedonthemarket(Regulation(EC)No258/97,concerningnovel
foodsandnovelfoodingredients). Undertheserules,approvalhasbeengivenfortheadditionof
plantsterolsinarangeoffoods,butthesearemostlyhigh-fatproductssuchasyellowfatspreads,
dairyproducts(e.g.,yogurt),mayonnaise,andsaladdressings. Unsaturatedformsofplantsterols,
phytostanols,havealsobeenaddedtofood. Anexamplehereofisanon-fattyalternative,chewable
plant stanol ester gum, for which efficacy has been confirmed recently [18]. Nevertheless, high
concentrationsofphytostanolsarenotnatural,ashighconcentrationsofphytostanolsareveryrarein
mostplants(withtheexemptionofafewcerealspeciesandtheirderivedproductssuchasriceoil)[7].
Natural water-soluble (glycosidic) plant sterols could be a good alternative for these (fatty)
phytostanol-andphytosterol-enrichedproducts[19],especiallyiftheycanbeobtainedfromeasyto
consumelow-fatandlow-energynaturalfoodsourcessuchasbeverages. Unfortunately,onlyvery
limiteddirect,quantitativedataisavailableonthenaturalpresenceof(glycosidic)plantsterols.Racette
etal.(2009)alreadynotedthatglycosylatedplantsterolsareoftenexcludedfromsterolanalysis,mostly
duetothelackofstandardsandanalyticaldifficulties[20]. Intheirstudy,totalplantsterolcontent,
includingglycosides,wascomputedindirectlyasthesumoftheindividualplantsterolsdetermined
by double (acidic and alkalic) hydrolysis. Their indirect analyses showed that glycosylated plant
sterols(ingeneral)comprised20%oftotalplantsterols,indifferentdiets. Theyalsosuggestedthat
nuts,seeds,legumes,wheatgerm,wholegrains,bran,fruit,andvegetablescouldbeimportantsources
ofglycosylatedplantsterols. ThepresenceofBSSGindietarysupplementsand(fatty)foodssuch
asnutsandwheathasalsobeentoucheduponbyPhillipsetal. (2005). Thehighestconcentrations
foundbackthen,usingindirectdetection,wereinflaxseedandsoybean(upto11mgper100gdry
weight,DW)[21]. Mulleretal. (2007)suggestedthatBSS(G)andergosterolfrombeercancompete
Nutrients2018,10,21 4of18
withcholesterolduringproteinbindingandassuchpreventcholesteroluptake. However,hedidnot
succeedinquantifyingthetrueamountsofBSS(G)presentinbeers[22].
Therefore,thismanuscriptaimedtoextendanextractionmethodandUPLC-MS/MSdetection
method, which was recently optimized and fully validated according to EC 2002/657 guidelines
andAssociationofAnalyticalChemists(AOAC)MScriteria,withthesetwocompoundsofinterest
(β-sitosterol-β-D-glucosideandergosterol)(MultipleReactionMonitoring,MRM)[23]. Campesterol,
stigmasterol, brassicasterol, ergosterol (provitamine D ), BSSG, and BSS concentrations were
2
determined in a broad range of plant-based beverages, including a variety of (concentrate-based)
juices,vegetablejuices,beers,teas,malt-based(non-alcoholic)drinks,andplant-basedmilkalternatives
(e.g.,oatorsoybeverages). Particularattentionwaspayedtosampleselectiontocoverarangeof
drinksthatisasbroadaspossible. Plantextract–containingsodaswerealsoincludedforcomparison.
Overallnutritionalvaluesandother(non-)beneficialcompoundsusedintheformulationwerealso
summarized(e.g.,concentrationsofproteins,vitamins,andmineralspresent).
2. MaterialsandMethods
Chloroform(analyticalgrade)andHPLCgrademethanol(MethanolOptima®)werepurchased
from FisherScientific (Leicestershire, UK). Methanol(analytical grade)was purchasedfromVWR
(Merck,Darmstadt,Germany). HPLCgrade,ultrapure(UP)waterwasacquiredfromanin-house
water purification system (Arium® 611UV, Sartorius Stedium Biotech, VWR, Haasrode, Belgium).
Cholesterol(≥99%,fromlanolin),β-sitosterol(BSS)(≥97%,fromsoybeans),brassicasterol(≥95%,
fromsemisynthetic),provitaminD (ergosterol)(≥97%,EuropeanPharmacopoeiaReferenceStandard),
2
andstigmasterol(≥97%,Supelco,CertifiedReferenceMaterial)werepurchasedfromSigmaAldrich
(St-Louis,Missouri,USA).Campesterol(≥98%,fromseedsofBrassicacampestris)wasobtainedfrom
WuhanChemFacesBiochemicalCo.,Ltd. (Wuhan,Hubei,China). β-sitosterol-β-D-glucoside(BSSG)
(≥95%,fromsemisynthetic)waspurchasedfromNeuroquest(Halifax,NS,Canada). Stocksolutionsof
eachcomponent(500or200ng/µL)anddilutionsupto1ng/µLweremadeinHPLCgrademethanol.
Allsolutionswerekeptat4◦Candprotectedfromdirectlight(brownflasksandadditionalaluminum
foilcoat).
Beverages were purchased from different suppliers/producers including Oat-ly AB (Mälmo,
Sweden), 2Food (Soesterberg, The Netherlands), Paulaner Brauerei GmbH & Co. KG (Münich,
Germany),ABInbev(Leuven,Belgium),OlgerdinEgillSkallagrímssonBrewery(Reykjavik,Iceland),
GhentUniversityCollege(Ghent,Belgium),TheCoca-ColaCompany(Ghent,Belgium),MelittaBelgië
n.v. (Lokeren,Belgium),DreamTM HainCelestialGroup,Inc. (Aalter,Belgium),ContinentalFoods
Belgium(Puurs,Belgium),PepsicoBeluxBVBA/SPRL(Zaventem,Belgium). DelhaizeLeLion/De
Leeuw(Brussel,Belgium),Alpro,TheWhiteWaveFoodsCompany(Wevelgem,Belgium),Forever
LivingProducts(Scottsdale,AZ,USA),Taofamily(Ternat,Belgium),NVBrasseriesAlken-MaesSA
(Malines/Opwijk, Belgium), Haacht Brewery plc (Boortmoorbeek, Belgium), Carlsberg Breweries
A/S(Copenhagen,Denmark),Palmbreweries(Steenhuffel,Belgium),BrasserieduBocq(Purnode,
Belgium),DuvelMoortgatNV(Puurs,Belgium),OmerVanderGhinsteBrewery(Bellegem,Belgium),
NestleSA(Vevey,Switzerland),andUnilever(Brussel,Belgium).
Statistical model designs were used to optimize the general analytical extraction procedure.
Dependent variables that might significantly affect the extraction efficiency were screened with
a fractional factorial D-optimal design. These variables were selected on the basis of a literature
surveyandfurtheroptimizationofonlytheinfluentialvariableswasperformedthroughresponse
surfacemodeling(RSM)(ModdePro12,Umetricssoftware,SartoriumStedimBiotech,Umeå,Sweden).
The optimal sample volume for liquid samples (beverages) was determined using an additional
small-scalefullfactorialdesign,and5mLwasfoundtobetheoptimalsamplevolume,bothinrelative
responsepermLandS/N[23](Table2).
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Table2. Summaryofthevalidatedextractionprotocoltoextractplantsterolsandergosterolfrom
adiverserangeofbeverages[23].
∆
1. 5mL(diluted) beverageina50mLtube
2. Additionofcholesterol(100µL,10ngor50ngperµL)
3. (Calibrationsamples:fortifiedwithdifferentplantsterols)
4. Liquid-liquidextractionwith8mLchloroform:methanol(2:1)
5. Vortex(30s)+ultrasonication*(10min)
6. Centrifugation(4400×g,10min)
7. Cottonwoolfilter
8. Secondliquid-liquidextraction(repeatstep2to5)
9. 1mLfreshchloroform:methanoladdedtothefilter(filterwashout)
10. Transfer2mLextracttonewtube
11. 15–20mindrying(underliquidN ,46◦C)
2
12. 180µLmethanol(vortex30s,ultrasonication*10min)
13. 20µLultrapureH O(vortex30s,ultrasonication*3min,vortex30s)
2
14. Centrifugation(12,300×g,10min)
15. Transfer150µLtoplasticLC-MSvialwithinsertforanalysis
∆
Samplesweredilutedifthefirstresultsshowedthatendogenousconcentrationsweretoohightobeableto
includecalibrationpointscontainingtwototentimestheendogenousconcentration(mostlyforBSSGandBSS);
*Ultrasonication:power100,frequency80kHz.
Anultra-highperformanceliquidchromatographytandemmassspectrometry(UPLC-MS/MS)
detectionmethodwasusedforthequantificationoffreeplantsterols,ergosterol,andBSSGinasingle
run. Previously,thismethodwasfullyvalidatedforquantificationofcampesterol,BSS,stigmasterol,
ergosterol,andbrassicasterol[23]. Preliminaryexperimentsshowedthatthismethodisalsosuitable
forquantificationofBSSG.SeparationwascarriedoutusinganAccelaTM HighSpeedLC(Thermo
FisherScientific,SanJose,CA,USA)equippedwithaThermoFisherScientific™HypersilGOLD™
C18Column(particlesize: 1.9µm,50×2.1mmI.D.). Themobilephasesusedwereultra-purewater
(solventA)andmethanol(LC-MSgrade,solventB).Allanalytescouldbeaccuratelyseparatedina
totalruntimeoflessthan10min(Table3). Thegradientstartedwithalineargradientof90%solvent
B(methanol)forthefirst2min,increasingto100%at5.5min,andthenheldat100%for1.5min(upto
7min). Afterward,thecolumnwasallowedtoequilibrateattheinitialconditionsof10%Aand90%B
for2min. Thedivertvalvewasusedtoloadthedetectorfrom1.0to4.5min. Schedulingwasusedto
increasesensitivity,bylimitingthedetectionwindowforeachanalyteto0.6minbeforeandafterthe
expectedretentiontime. DetectionwascarriedoutonaTSQVantagetriplestagequadrupolemass
spectrometerequippedwithanatmosphericpressurechemicalionizationprobe(APCI)(ThermoFisher
Scientific,SanJose,CA,USA).Injectionvolumeswere10µLeachandtheAPCIsourcewasoperated
inthepositiveionmode. Thedischargecurrentwassetat±4µA.Thesheath,sweepandauxiliarygas
pressuresweresetat20,2,and10arbitraryunits,respectively,thecapillarytemperatureat300◦C,
andthevaporizertemperatureat320◦C.Thecollisiongaspressurewaskeptat1.5mTorr,andthe
cycletimewas0.8s. Datawereacquiredintheselected/multiplereaction-monitoring(SRM/MRM)
mode. Allspecifiedproductions(Table3)wereusedforpeakintegration,ionratiodeterminations,
andquantificationpurposes.
Nutrients2018,10,21 6of18
Table3.Selected/multiplereactionmonitoring(SRM/MRM)specificsforallcompoundsofinterest:
precursor ions, product ions (as m/z, mass over charge), absolute and relative retention time
(RT,inminutes,min),appropriateS-Lensamplitude(volt,V),andthecorrespondingcollisionenergy
(CE,inelectronvolt,eV).
MeanRelativeIon RetentionTime
PrecursorIon ProductIons S-LensVoltage CollisionEnergy
Analyte Abundancy* (Relative)
(m/z) (m/z) (%) (min) (V) (eV)
91 70 47
β-Sitosterol-β-D-glucoside 397.3 95 73 1.80 88 35
BSSG 105 100 (0.77) 40
147 93 28
69 78 23
Ergosterol 91 100 2.04 53
379.3 120
ProvitamineD2 105 90 (0.87) 34
15 82 24
105 100 43
159 67 2.27 23
Brassicasterol 381.3 82
297 93 (0.97) 14
311 40 13
91 83 52
Cholesterol 2.35
369.3 95 69 84 34
internalstandard (1.00)
105 100 40
81 67 35
91 85 2.61 49
Campesterol 383.3 86
95 74 (1.11) 34
105 100 43
81 64 37
91 91 2.63 52
Stigmasterol 395.3 59
105 100 (1.12) 44
297 90 18
91 70 47
β-sitosterol 95 73 2.90 35
397.3 88
BSS 105 100 (1.23) 40
147 93 28
*Insolvent,atfullwidthhalfmaximum(FWHM)andrelativetotheproductionwiththehighestintensity.
Arearatioswerecalculatedrelativetotheinternalstandard(ISTD)cholesterol,whichwasadded
to both calibration and unknown samples, to compensate for losses during sample preparation
and/orvariationoftheanalyticalanalysis. Cholesterolwasconsideredasuitableinternalstandard
as no significant endogenous concentrations are present in the samples of interest (plant-based)
andcholesterolisverysimilartothecalibratedanalytes(Figure1; Tables1and3),chemicallyand
in retention time but nevertheless chromatographically distinguishable and less expensive than
isotopicallylabeledstandards. Applyingthismethodtoothersamplesthat,contrarytothesamples
analyzedinthecurrentstudy,docontainsignificantconcentrationsofendogenouscholesterolwould
imply the use of another internal standard (e.g., 5α-cholestan-7β-ol or a deuterated (glycosidic)
plantsterol).
Ofeachbeverage,atleastthreenon-fortifiedsampleswereextractedtogetherwithanine-point
matrix-matchedcalibrationcurve(≥12samplespermatrix),constructedbaseduponninefortification
levels (0, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 times the minimal expected endogenous concentration of
eachplantsterolindividually). Theminimalexpectedendogenousconcentrationwaspreliminary
determined based upon calculated expected endogenous concentrations and standard addition
(analysisofmatrixmatchedsampleswithknownaddedconcentrationsofplantsterols). Calculations
combinedavailablereferencevaluesforsolidingredientswiththeirexpectedminimalcontribution
(%)tothedifferentbeverages. Allsampleswereruntwice,totakeintoaccountanalyticalvariance,
andmean(n=6)±standarddeviationoftheseduplicaterunsarereportedinTables4and5.
Nutrients 2017, 10, 21   Nutrients 2017, 10, 21   7 of 18  7 of 18 
Table  4.  UPLC‐MS/MS  determined  concentrations  of  BSS,  BSSG,  stigmasteTroabl, lec a4m. pUesPteLrCo‐lM,  S/MS  determined  concentrations  of  BSS,  BSSG,  stigmasterol,  campesterol, 
brassicasterol, and ergosterol (provitamin D2) in a diverse range of beverages (fruibtr jausisciecsa,s vteergoelt, aabnled  ergosterol (provitamin D2) in a diverse range of beverages (fruit juices, vegetable 
juices, plant‐based milk alternatives, gel, sodas, teas, and (non‐alcoholic) malt‐jbuaisceeds,  dprlainnkt‐sb aasnedd  milk alternatives, gel, sodas, teas, and (non‐alcoholic) malt‐based drinks and 
beers).  beers). 
mg per 100 mL  μg per 100 mL  mg per 100 mL  μg per 100 mL 
Category  Product Name  BSS  BSSG  Brassicasterol  Campesterol  CSatitgegmoarsyt erol  ErgosPtreordolu ct N ame  BSS  BSSG  Brassicasterol  Campesterol  Stigmasterol  Ergosterol   
Apple juice  0.21 ± 0.01  4.1 ± 1.2 *  NF (<0.75)  27 ± 3  2.6 ± 0.4  Apple juNicDe      0.21 ± 0.01  4.1 ± 1.2 *  NF (<0.75)  27 ± 3  2.6 ± 0.4  ND    
Orange juice  0.42 ± 0.09  8.3 ± 2.3 *  NF (<1.5)  71 ± 12  23 ± 3  Orange jNuiDce      0.42 ± 0.09  8.3 ± 2.3 *  NF (<1.5)  71 ± 12  23 ± 3  ND    
Fruit juices  Pomegranate juice  2.1 ± 0.3  32 ± 7  NF (<3)  139 ± 18  Fruit ju<i3c0e s  Pomegr1a7n ±a t6e  juice   2.1 ± 0.3  32 ± 7  NF (<3)  139 ± 18  <30  17 ± 6   
Multifruit‐carrot juice  2.5 ± 0.2  16 ± 3  NF (<3)  607 ± 12  224 ± 10  MultifruNit‐Dca  rrot juic e  2.5 ± 0.2  16 ± 3  NF (<3)  607 ± 12  224 ± 10  ND    
Fresh orange‐banana juice  5.3 ± 2.2  >90  NF (<3)  846 ± 93  610 ± 35  Fresh oNrFan (g<3e)‐ banan a juice  5.3 ± 2.2  >90  NF (<3)  846 ± 93  610 ± 35  NF (<3)   
Tomato juice  0.36 ± 0.02  4.4 ± 0.5  NF (<2)  155 ± 10  331 ± 27  Tomato NjuDic e     0.36 ± 0.02  4.4 ± 0.5  NF (<2)  155 ± 10  331 ± 27  ND    
Nutrients2018,10,21 7of18
Mixed vegetable juice (a)  0.74 ± 0.05  6.2 ± 0.5  NF (<3)  242 ± 24  596 ± 64  Mixed vNegDet a ble juic e (a)  0.74 ± 0.05  6.2 ± 0.5  NF (<3)  242 ± 24  596 ± 64  ND    
Vegetable  Vegetable 
Mixed vegetable juice (b)  0.72 ± 0.10  12 ± 3  NF (<3.75)  177 ± 25  359 ± 72  Mixed v29eg ±e 4ta ble juic e (b)  0.72 ± 0.10  12 ± 3  NF (<3.75)  177 ± 25  359 ± 72  29 ± 4   
juices  juices 
Table4. UPLC-MS/BMeetrSoodt jeuitcee rminedconcentr0a.4t2i o± n0.0s7 ofB7S.3S ± ,1.B2 SSG,sNtFi g(<m3) asterol,47c ±a 8m  pester4o0l ±, 3b raBsesetircoaNotsF jt u(e<ic3re)o  l,an  dergostero0l.4(2p ± r0o.0v7 itam7.3i n± 1D.2 2)inaNFd (i<v3)e rseran4g7 e± 8o f 40 ± 3  NF (<3)   
beverages(fruitjuicCeasr,rovt ejugicee tablejuices,plant-b2a.7s ±e 0d.4 milk1a8 l±t e4 rnativeNsF, (g<3e)l ,sodas67,7t ±e a68s ,and1(2n70o ±n 6-5a lCcaorrhootN jluFii cc(e<) 3m)  alt- baseddrinks2.a7n ± d0.4b eers)1.8 ± 4  NF (<3)  677 ± 68  1270 ± 65  NF (<3)   
Coconut‐rice  0.51 ± 0.07  2.8 ± 0.9  NF (<3)  72 ± 10  76 ± 13  CoconutN‐rDic e    0.51 ± 0.07  2.8 ± 0.9  NF (<3)  72 ± 10  76 ± 13  ND   
Category Rice  ProductName 1.4 ± 0.1  2.4 ± 0m.6g per100m10L ± 3  260 ± 28  234 ± 23  Rice  ND    µgper100m1L.4 ± 0.1  2.4 ± 0.6  10 ± 3  260 ± 28  234 ± 23  ND   
Plant‐based  SAopyp lejuice 2.5 ± 0.5  0.21B4±.S9S 0±. 021.1  4.6 ± 04.4.1B ±SS1G.2*1290 ± 291  BPrlNaasFns9ti(9‐c<b8a0a s±.s7t ee15rd1)o1 l Soy  ND Cam27pe± st3erol 2.5S ±ti2 g0.6.m5± as0te.4rol4.9 ± 2.1  4E.6r g±No 0sD.t4e rol 1290 ± 291  998 ± 111  ND   
COarsahnegwe juice 2.7 ± 0.4  0.42±>60.00 9 NF (<83.)3 ±2.3* 279 ± 44  NF(1<51 ±.5 1)  CashewN F (<3)  71± 12 2.7 ± 02.34 ±3 >60  NF (N<3D)  279 ± 44  15 ± 1  NF (<3)   
Fruitjuicesmilk  Pomegranatejuice 2.1±0.3 32±7 NmFil(<k3 ) 139±18 <30 17±6
AMlmulotinfrdu (ita-)c aurnrrootajustiecde  2.6 ± 0.6  2.57±8 0±. 214  NF (<3)1 6±3 101 ± 30  NF(<<330)  AlmondN (aD)  unr6o0a7st±e d1 2 2.6 ±2 02.46 ±10 78 ± 14  NF (N<3D)  101 ± 30  <30  ND   
alternatives  AFTolrmemsohantoodr ja(ubni)gc eero-baasnteadn ajuice 2.5 ± 0.1  05.3.36±±13 20±..20 22  NF (<24). 4>±900.5 62 ± 4  alteNN1rFFn91a((5<<ti 23±v)) e1s0 9 AlmondN (bD)  roa81st45e65d±±   1903 2.5 ±36 013.101 ±±2375 13 ± 2  NNF F(N<(2D<) 3) 62 ± 4  1915 ± 109  ND   
OMaitx (ead) vegetablejuice(a) 2.1 ± 0.2  0.74±26 0±.0 45  NF (<36). 2±0.5 475 ± 30  NF18(2< 3±) 16  Oat (a) ND  242±  24 2.1 ±5 09.62 ±64 26 ± 4  NF (N<3D)  475 ± 30  182 ± 16  ND   
Vegetablejuices Mixedvegetablejuice(b) 0.72±0.10 12±3 NF(<3.75) 177±25 359±72 29±4
OBaete t(rbo)o tjuice 3.9 ± 0.7  0.42±33 0±.0 47  217 ± 172. 3±1.2 1098 ± 61  NF(<<135)  Oat (b)N F (<3)  47±  8 3.9 ± 04.07 ±3 33 ± 4  21N7 F± 1(<23 ) 1098 ± 61  <15  NF (<3)   
Gel  ACClaooercr ovonetrujaut -girceiecl ebeverage  0.22 ± 0.03  02.5.71±±17 00±..40 57  NF (<0.7251.8)8 ±±04.9 23 ± 5  NNGFF2.e((2<<l  ±33 ))0.9  Aloe veraN gDel  bev67e72r7a±±g e16 08 0.22 ±1 72067.00±3± 1635 17 ± 5  NF N(<FN0.(D7<53))  23 ± 5  2.2 ± 0.9  ND   
LReimceonade (a) (orange)  0.48 ± 0.04  1.41.±5 ±0 .01.3*  NF (<0.725.4) ±0.6 73 ± 3  101±9 3± 2  LemoNnFa d(<e0 (.a7)5 )( o2r6a0n±g e2) 8 0.48 ±2 03.404± 231.5 ± 0.3*  NF (<N0.D75)  73 ± 3  19 ± 2  NF (<0.75)   
Soy 2.5±0.5 4.9±2.1 4.6±0.4 1290±291 998±111 ND
BCioaslehmewonade (a) (elderberry)  0.19 ± 0.02  2.72±.3 0±. 40.7  NF (<1.5) >60 24 ± 2  NF5(2< ±3 )15  BioleNmFo n(<a0d.e7 5()a 2) 7(e9l±d e4rb4erry)  0.19 ± 01.502± 1 2.3 ± 0.7  NFN (<F1(.<53) ) 24 ± 2  52 ± 15  NF (<0.75)   
Plant-basedmilkalteSrondaatisv es BAAiollmmleoomnnoddn((aabd))eur o(nbar)so t(aegsditnedger‐orange)  0.17 ± 0.05  22..652±±.5 00±.. 610.7  NF (<0.77518)3 ±±124 24 ± 3  NNSoFF4d.((3a<< ±s23  ))1.0  BioleNmFo n(<a0d.e7 5()b 1) 60(21gi±±n g34e0r‐orange)  0.17 1±9 01.5<053± 01092.5 ± 0.7  NF (<NN0.DD75)  24 ± 3  4.3 ± 1.0  NF (<0.75)   
SOoadta( aw)ith plant extract (a)  0.05 ± 0.01  2.11±.1 0±. 20.2  NF (<0.752)6 ±4 8.0 ± 1.7  NF1.(3< ±3 )0.3  SodaN wFi t(h< 0p.l7a5n) t4 e7x5t±r ac3t0 (a)  0.05 ±1 08.201± 16 1.1 ± 0.2  NF (<N0.D75)  8.0 ± 1.7  1.3 ± 0.3  NF (<0.75)   
Oat(b) 3.9±0.7 33±4 217±12 1098±61 <15 NF(<3)
Gel SAoldoea vweriathg pellabnetv eexratrgaect (b) (stevia)  0.05 ± 0.01  0.202.6±0 0±. 003.10  NF (<0.751)7 ±5 7.3 ± 1.1  NF1(<.90 ±.7 05.)3  SodaN wFi t(h< 0p.l7a5n) t e2x3t±r ac5t (b) (stevia)  0.05 ±2 0.2.0±1 0.90.60 ± 0.10  NF (<N0.D75)  7.3 ± 1.1  1.9 ± 0.3  NF (<0.75)   
SLBoeidomlae owmniaotdnhea pd(laea)n((ato )er(xaentlrdgaeecr)tb (ecr)r (yp)each)  0.06 ± 0.01  00..41891±±.5 00±.. 00042.5  NF (<0.172.5.53) ±±00..37* 5.8 ± 1.2  NNF0F.(8(<<00 1±..7 505).)31 SodaN wFi t(h< 0p.l7a5n) t e27x43t±±r ac32t (c) (peach)  0.06 ± 5012.90±1± 125 1.5 ± 0.5  NNNF FF(<((0<<.7005..77) 55)) 5.8 ± 1.2  0.80 ± 0.31  NF (<0.75)   
TBeiao lienmfuosnioadn e(a(b) )(ginger-orange) 0.08 ± 0.01  0.107.6±7 0±. 005.12  NF (<0.725.5) ±0.7 9.2 ± 1.7  NF1(<.10 ±.7 05.)3  Tea iNnfFu s(i<o0n.7 (5a))  24±  3 0.08 ±4 0.3.0±1 1.00.67 ± 0.12  NNF F(<(0<.705.7) 5) 9.2 ± 1.7  1.1 ± 0.3  NF (<0.75)   
Sodawithplantextract(a) 0.05±0.01 1.1±0.2 NF(<0.75) 8.0±1.7 1.3±0.3 NF(<0.75)
Sodas TSeoad ianwfuisthiopnl (abn)t extract(b)(stevia) 0.06 ± 0.01  0.005.6±5 0±. 001.17  NF (<00.7.650) ±0.10 9.6 ± 1.3  NF1(<.50 ±.7 05.)2  Tea iNnfFu s(i<o0n.7 (5b)) 7 .3± 1.1 0.06 ±1 0.9.0±1 0.30.65 ± 0.17  NNF F(<(0<.705.7) 5) 9.6 ± 1.3  1.5 ± 0.2  NF (<0.75)   
Teas  TSeoad ianwfuisthiopnl (acn)t extract(c)(peach) 0.06 ± 0.01  0.006.6±8 0±. 001.12  NF (<0.715.5) ±0.5 6.9 ± 0.8  NFT2(e<.a30s ±. 7 05.)2  Tea iNnfFu s(i<o0n.7 (5c)) 5 .8± 1.2 0.06 0±. 800.0±1 0.310.68 ± 0.12  NNF F(<(0<.705.7) 5) 6.9 ± 0.8  2.3 ± 0.2  NF (<0.75)   
Teainfusion(a) 0.08±0.01 0.67±0.12 NF(<0.75) 9.2±1.7 1.1±0.3 NF(<0.75)
ITceead itnefau (sbio) n(b) 0.06 ± 0.01  0.006.9±3 0±. 001.32  NF (<00.7.655) ±0.17 8.3 ± 1.6  NF5(<.80 ±.7 05.)8  Iced tNeaF  ((b<)0 .75) 9.6± 1.3 0.06 ±1 0.5.0±1 0.20.93 ± 0.32  NNF F(<(0<.705.7) 5) 8.3 ± 1.6  5.8 ± 0.8  NF (<0.75)   
Teainfusion(c) 0.06±0.01 0.68±0.12 NF(<0.75) 6.9±0.8 2.3±0.2 NF(<0.75)
Teas IIcceedd tteeaa ((cb))  0.05 ± 0.01  0.006.8±4 0±. 001.08  NF (<00.7.953) ±0.32 6.7 ± 0.8  NF9(<.50 ±.7 15.)4  Iced tNeaF  ((c<)0 .75) 8.3± 1.6 0.05 ±5 0.8.0±1 0.80.84 ± 0.08  NNF F(<(0<.705.7) 5) 6.7 ± 0.8  9.5 ± 1.4  NF (<0.75)   
* UsingI cae dgtreao(uc)p specific calibration curve f0r.0o5m± 0r.e01spectively p0.o84m±e0g.0r8anate juice N(fFr(*u< 0iUt.7 s5ju)inicge sa)  gorro luapg esrp6 .e7c±if0i.c8 calibration cu9r.5v±e 1f.r4om respectiveNlyF (p<0o.7m5)egranate juice (fruit juices) or lager 
*Usingagrou(pa)s p(ebceiefircs)c;a libr aIntidonicactuersv ea fprolamntr‐ebsapseecdti vbeelvyeproagmee gthraant actaenj ubicee c(ofrnusiitdjeuriceeds )ao grolaogde rs(o(aau)) r((cbbeee eeorrfss ))f;;r ee  aIInnnddd iiccaatteess aap plalannt-tb‐absaesdedb ebveevreargaegteh athtacat ncabne cboen csoidnesriedderaegdo ao dgood source of free and 
sourceoffreeacnodncjuognajutgedat epdlpanlatn tssteterroollss;;   IInnddicicaatetessa pal apnlta-bnat‐sbedasbeedv ebraegveertahgaet cothnatat incsoonntalyincmso onodjuneglryaa tteemd(yo edplleloarwantt)e o srtelorwolsc;o ncentr aItniodnisca(oteras nga e)polafnptl‐abnatssetder oblse.vNerFa,gneo ttfhoautn dc;ontains only moderate 
ND,notdeterm(yineeldlo.w) or low concentrations (orange) of plant sterols. NF, not found; ND, not de(tyeermlloinwe)d o r low concentrations (orange) of plant sterols. NF, not found; ND, not determined
Nutrients2018,10,21 8of18
Nutrients 2017, 10, 21   8 of 18 
Table  5.  UPLC‐MS/MS  determined  concentrations  of  BSS,  BSSG,  stigmasterol,  campesterol, 
Table5. UPLC-MS/MSdeterminedconcentrationsofBSS,BSSG,stigmasterol,campesterol,brassicasterol,andergosterol(provitaminD )inadiverserangeof
2
beverages(fruitjuices,vegetablejuices,plant-basedmilkalternatives,gel,sodas,teas,baransdsi(cnaostne-raollc, oahnodl iecr)gmosateltr-obla (sperdovdirtainmkisn aDn2d) ibne ae rdsi)v.erse range of beverages (fruit juices, vegetable 
juices, plant‐based milk alternatives, gel, sodas, teas, and (non‐alcoholic) malt‐based drinks and 
beers). 
mgper100mL µgper100mL
Category ProductName
BSS BSSG Brassicasterol Campesterol Stigmmasgt epreorl 100 mL Ergosterol μg per 100 mL 
Chinesemaltdrink 0.07±0.02 0.95±0.33 CNatFeg(o<r0y. 75) Pro6d.u6c±t n2a.m1e  <LOQ(<2) ND  
BSS  BSSG  Brassicasterol  Campesterol  Stigmasterol  Ergosterol 
Icelandicmaltdrink 0.14±0.04 2.74±1.31 NF(<1.5) 19±4 2.4±0.9 ND
Non-alcoholic Non-alcoholiclager(a) 0.04±0.01 0.50±0.07 NF(<0.75) Chinese m6a.l8t d±rin2k.4 01..007 ±± 00.0.42 0.95 ± 0N.3F3 (<0.75N)F (<0.75) 6.6 ± 2.1 <LOQ (<2) ND  
maltdrinks Icelandic malt drink 0.14 ± 0.04 2.74 ± 1.31 NF (<1.5) 19 ± 4 2.4 ± 0.9 ND  
Non-alcoholiclager(b) 0.07±0.03 1.6±0.5 NNFon(<‐ 0.75) 11±3 1.9±0.8 ND
Non-alcoholic lager (a) 0.04 ± 0.01 0.50 ± 0.07 NF (<0.75) 6.8 ± 2.4 1.0 ± 0.4 NF (<0.75)  
Non-alcoholicwheatbeer(a) 0.07±0.02 1.0±0.1 alcoNhoFli(c< m0.a7l5t ) 7.7±1.9 0.88±0.35 ND
Non-alcoholic lager (b) 0.07 ± 0.03 1.6 ± 0.5 NF (<0.75) 11 ± 3 1.9 ± 0.8 ND  
Non-alcoholicwheatbeer(b) 0.12±0.03 1.4±0.2* NdrFin(k<s0 .75) Non-alcoho1li4c w±h3eat beer (a) 02..017 ±± 00.0.42 1.0 ± 0.1 ND NF (<0.75) 7.7 ± 1.9 0.88 ± 0.35 ND  
Lager(a) 0.20±0.04 2.2±0.3 NF(<0.75) Non-alcoho2li4c w±h5eat beer (b) 0.132 ±± 01.03 1.4 ± 0N.2 F* (<0.75N)F (<0.75) 14 ± 3 2.1 ± 0.4 ND  
Lager(b) 0.26±0.02 1.7±0.3* NF(<0.75) Lager (a) 31±4 0.250 ±± 01.04 2.2 ± 0.3 ND NF (<0.75) 24 ± 5 3 ± 1 NF (<0.75)  
Lager(c) 0.25±0.04 1.2±0.3* NF(<0.75) Lager (b) 39±5 0.276 ±± 01.02 1.7 ± 0.3 * ND NF (<0.75) 31 ± 4 5 ± 1 ND  
Lager (c) 0.25 ± 0.04 1.2 ± 0.3 * NF (<0.75) 39 ± 5 7 ± 1 ND  
Lager(d) 0.23±0.03 1.7±0.3* NF(<0.75) 23±4 5±1 ND
Lager (d) 0.23 ± 0.03 1.7 ± 0.3 * NF (<0.75) 23 ± 4 5 ± 1 ND  
Wheatbeer(a) 0.28±0.04 2.8±0.4* NF(<0.75) 52±3 6±1 4.1±0.4
Wheat beer (a) 0.28 ± 0.04 2.8 ± 0.4 * NF (<0.75) 52 ± 3 6 ± 1 4.1 ± 0.4  
Beers Wheatbeer(b) 0.38±0.09 3.2±0.6* NF(<0.75) Wheat beer5 (3b)± 11 0.378 ±± 02.09 3.2 ± 0.6 * ND NF (<0.75) 53 ± 11 7 ± 2 ND  
Wheatbeer(c) 0.26±0.02 1.9±0.3* NBFee(r<s 0.75) Wheat beer 3(c0) ±2 0.236 ±± 01.02 1.9 ± 0.3 * ND NF (<0.75) 30 ± 2 3 ± 1 ND  
Wheatbeer(d) 0.27±0.04 3.4±0.4* NF(<0.75) Wheat beer 3(d7)± 6 0.257 ±± 01.04 3.4 ± 0.4 * ND NF (<0.75) 37 ± 6 5 ± 1 ND  
Ale(bottlefermented)(a) 0.37±0.05 2.0±0.3* NF(<0.75) Ale (bottle f4e9rm±en7ted) (a) 0.397 ±± 01.05 2.0 ± 00.3.8 *0 ±0.1N8F (<0.75) 49 ± 7 9 ± 1 0.80 ± 0.18  
Ale (bottle fermented) (b) 0.23 ± 0.03 4.5 ± 0.5 * NF (<0.75) 36 ± 4 6 ± 1 6.0 ± 0.3  
Ale(bottlefermented)(b) 0.23±0.03 4.5±0.5* NF(<0.75) 36±4 6±1 6.0±0.3
Ale (bottle fermented) (c) 0.25 ± 0.03 2.5 ± 0.5 * NF (<0.75) 36 ± 4 4 ± 1 ND  
Ale(bottlefermented)(c) 0.25±0.03 2.5±0.5* NF(<0.75) 36±4 4±1 ND
Ale (bottle fermented) (d) 0.23 ± 0.03 1.3 ± 0.1 * NF (<0.75) 7 ± 2 2 ± 1 4.6 ± 0.8  
Ale(bottlefermented)(d) 0.23±0.03 1.3±0.1* NF(<0.75) 7±2 2±1 4.6±0.8
Ale (bottle fermented) (e) 0.09 ± 0.02 0.9 ± 0.1 * NF (<0.75) 36 ± 4 6 ± 1 ND  
Ale(bottlefermented)(e) 0.09±0.02 0.9±0.1* N*F U(<s0i.n75g) a group 3s6pe±ci4fic calibratio6n± c1urve from resNpDectively pomegranate juice (fruit juices) or lager 
*Usingagroupspecificcalibrationcurvefromrespectivelypomegranatejuice(fruitjuices)orlage(ra)( a)(b(beeeerrss)); ;  InInddiciactaetsesa pal apntl-abnats‐e dbabseevder abgeevtehraatgceo ntthaaint scoonnlytaminosd eornaltye (ymeolldower)aoter (yellow) or low 
lowconcentrations(orange)ofplantsterols.NF,notfound;ND,notdetermined. concentrations (orange) of plant sterols. NF, not found; ND, not determined. 
For almost half of the samples (n = 19/49), endogenous concentrations of BSSG and/or BSS 
turned out to be too high to be able to include fortified samples with two to ten times the 
endogenous concentrations. For those matrices, additional calibration curves were made in diluted 
samples and additional diluted non‐fortified samples were analyzed (diluted 4‐ to 30‐fold with UP 
water, depending on the matrix). For all samples, endogenous plant sterol concentrations were 
determined by fitting the compounds’ area ratio of non‐fortified samples into the corresponding 
calibration curve. For diluted samples, the concentrations in undiluted samples were recalculated 
afterward. 
3. Results 
3.1. Data Analysis and Quality Assurance of the Analytical Method: Limits of Detection and Quantification 
Lower limits of quantification (LLOQs) in solvent (90:10 methanol:H2O) for the method used 
were between 0.5 and 1.5 ng per mL. In (diluted) beverages, the LLOQ was 0.5–3.0 μg per 100 mL for 
liquid samples, depending on the general composition of the sample and the compound of interest. 
In general, fatty and protein rich beverages (e.g., soy and oat beverage) hampered the detection of 
very low concentrations of plant sterols. Fortunately, most of these beverages were also relatively 
high in plant sterols. Previous results showed that the limits of detection (LODs) for solid matrices 
for the different compounds were between 10 and 30 μg per 100 g and LLOQs were between 20 and 
100 μg per 100 g [23].
Nutrients2018,10,21 9of18
For almost half of the samples (n = 19/49), endogenous concentrations of BSSG and/or BSS
turnedouttobetoohightobeabletoincludefortifiedsampleswithtwototentimestheendogenous
concentrations. Forthosematrices,additionalcalibrationcurvesweremadeindilutedsamplesand
additionaldilutednon-fortifiedsampleswereanalyzed(diluted4-to30-foldwithUPwater,depending
onthematrix). Forallsamples,endogenousplantsterolconcentrationsweredeterminedbyfittingthe
compounds’arearatioofnon-fortifiedsamplesintothecorrespondingcalibrationcurve. Fordiluted
samples,theconcentrationsinundilutedsampleswererecalculatedafterward.
3. Results
3.1. DataAnalysisandQualityAssuranceoftheAnalyticalMethod: LimitsofDetectionandQuantification
Lowerlimitsofquantification(LLOQs)insolvent(90:10methanol:H O)forthemethodused
2
werebetween0.5and1.5ngpermL.In(diluted)beverages,theLLOQwas0.5–3.0µgper100mLfor
liquidsamples,dependingonthegeneralcompositionofthesampleandthecompoundofinterest.
Ingeneral,fattyandproteinrichbeverages(e.g.,soyandoatbeverage)hamperedthedetectionofvery
lowconcentrationsofplantsterols. Fortunately,mostofthesebeverageswerealsorelativelyhighin
plantsterols. Previousresultsshowedthatthelimitsofdetection(LODs)forsolidmatricesforthe
differentcompoundswerebetween10and30µgper100gandLLOQswerebetween20and100µg
per100g[23].
3.2. QuantificationofCholesterol-Lowering(Conjugated)PlantSterolsandErgosterol(ProvitaminD )with
2
UPLC-MS/MS
All UPLC-MS/MS determined concentrations of BSS, BSSG, stigmasterol campesterol,
brassicasterol,andergosterol(provitaminD )inadiverserangeofbeverages(fruitjuices,vegetable
2
juices,plant-basedmilkalternatives,gel,softdrinks,teas,(non-alcoholic)malt-baseddrinks,andbeers)
havebeensummarizedinTables4and5.
4. Discussion
Wherepossible,obtainedplantsterolconcentrationswerecomparedtopreviouslyobtainedthin
layerchromatography(TLC),gas-orliquidchromatography—massspectrometry(LC/GC-MS)and
GC-FID(FlameIonizationDetector)resultsavailableinliterature.
4.1. FruitJuices
BSSGwaspresentinveryhighconcentrations(upto>90mgper100mL).Theseconcentrations
aremuchhigherthanthemeanconcentrationsdeterminedincorrespondingplantsinthepast,through
indirectanalysis. Theseresultsshowedthatingeneralonly20%oftheplantsterolsfoundinedible
plants(<10mgper100mL)wereconjugated[20]. Itisthusverylikelythatglucose-conjugatedplant
sterols are currently being underestimated in solid matrices, where they are tightly matrix-bound.
However,duetotheirwater-solublenature,theyareenrichedthroughouttheproductionprocessof
plant-basedbeverages. Inaddition,itcan’tbeexcludedthatthepreviouslyusedindirectmethodbased
uponchemicalhydrolysis[20]wasnotsufficienttoreleaseconjugatedplantsterolsfromthematrix
andcompletehydrolysisoftheβ-glycosidicboundatthesametime.
BSSwasthemostabundantfreeplantsterolfound,inlinewithmeanfreeplantconcentrationsin
higherplants,whereBSSaccountedfor50to80%ofthetotalamountofplantsterols[24]. Thehighest
concentration of BSS (5.3 ± 2.2 mg per 100 mL) was measured in the fresh orange-banana juice.
Asacomparison,thisis2.1and2.5timeshigherthantheconcentrationofBSSinthepomegranate
juiceandthemultifruit-carrotjuice,respectively. ThelowestconcentrationofBSSwasfoundinthe
concentratedorangeandapplejuice(13and25timelower,respectively).
Interestingly,thetwofruitjuicesthatcontainedorangejuiceasamainingredient(80%infresh
orange-bananajuice,100%intheconcentrate-basedorangejuice;SupplementaryMaterialsTableS1)
Nutrients2018,10,21 10of18
containedverysimilarratiosofBSSG:BSS:campesterol,butthefreshorange-bananajuicehad12to
14timeshigherconcentrations. Theconcentrationdifferenceforstigmasterolwas26-fold,butthatcan
beadirectconsequenceofthe20%bananacontent. Bananasaretypicallyveryhighinstigmasterol;
containingupto200mgper100gdryweightofbananapseudostem(pulp)[25–27].
Thelargedifferencebetweenbothorangejuicebasedbeveragescanbeexplainedbylookinginto
detailattheresultsobtainedfortheappleandorangeconcentratebasedjuices,whichhappenedtobe
producedbythesameproducer. Theirplantsterolconcentrationswerefoundtobeinlinearcorrelation
withthereportedconcentrationsinthecorrespondingfruits(orange23–24mgandapple12mgper
100gfreshweightwith11mgBSS,0.3mgcampesteroland0.05mgstigmasterol)[23,28–31]. Forboth
juices(100%fruit),onlyaround2%oftheinitialplantsterolcontentofthefruitsseemedtoendupin
theconcentrate-basedjuices(gtomL),whichsuggeststheyusedsimilarproductionprocessesforboth
concentrate-basedjuices,whowereunfortunatelyequallyincapableofcontainingplantsterolsintothe
finaljuices. Thisillustratesthepossiblelossofbeneficialcompoundsduringtheconcentrationprocess.
Previousresearchhasshownthattheindustrialproductionprocesscanhaveasignificantinfluenceon
thefinalconcentrations(e.g.,vitaminCandphenols). Previousresearchalsoshowedthatcommercial
squeezingoforangesextracted22%morephenolsthanhandsqueezing,however,thefreezingprocess
caused a dramatic decrease in phenols and the concentration process caused a mild precipitation
ofthesecompoundstothejuicecloud[32]. Similarresultshavebeenreportedforphenolsinapple
juices,butthiscouldbepreventedbyusinginitialhightemperature-shorttime(HTST)treatmentand
diffusionextractioninsteadofpressing[33]. Additionalresearchwillbeneededtoelucidatetheexact
influenceofdifferentfruitjuiceconcentrateproductionprocessesontheconcentrationsofplantsterols
inthefinaljuice.
Inpomegranatejuiceanotherpeculiarresultwasdetectedinthechromatogramforstigmasterol.
Stigmasterol could not be quantified as an intense interfering peak appeared at a retention time
veryclosetotheretentiontimeofstigmasterol(withthesameprecursorandfragmentionmasses).
Uponadditionofstigmasterol(matrix-matchedcalibrationcurve),itwasconfirmedthatthispeak
wasnotstigmasterol. Itseemsthatpomegranate(juice)containsaspecificcompound/plantsterol,
very similar to stigmasterol, or a specific conjugated form of stigmasterol that deconjugates upon
ionization (see also 4.6 Other water-soluble glucose-conjugated plant sterols). This illustrates the
difficultiescausedbythelackofanalyticalstandardsfor(conjugated)plantsterols. Over200different
types of plant sterols have been described [7], but for only a very limited number of those plant
sterolsanalyticalgrade(≥95%purity)standardsareavailable. Assuch,itisverylikelythatanalytical
resultsstillunderestimatethecontributionof,mostlyexotic,novelfruitsandotherpartsofplantsthat
werepreviouslynotusedforconsumption(novelfoodslistEU1997,e.g.,pomegranate,chiaseeds,
Aloevera).EspeciallyasarevisionofthenovelfoodsdirectiveisduetocomeintoforceinJanuary2018;
theapprovalprocesswillbesignificantlyshortenedandsimplified(andthuslessexpensive)fornew
fruitsorjuicesaslongasa25yearshistoryofusecanbeshowninthecountryoforigin(newDirective
forTraditionalFoods,ECRegulationNo. 2015/2283). Theintroductionofthesenewfruitsorjuices
ontothemarketwillhampertheaccuratedetectionofplantsterolsevenmore, asthesefruitswill
beaccompaniedbylesswellknownplantsterols, whereforenoanalyticalstandardsareavailable.
Additionalresearchwillalsobeneededtoevaluateiftheselesswell-knownplantsterolsexhibitthe
samehealthbenefits.
The tropical multifruit-carrot juice was also concentrate-based (18% orange, 19% pine apple,
5%carrotand3%otherfruitsincludingpassionfruit,SupplementaryMaterialsTableS1). Orange,pine
appleandcarrotsaremoderatelyhighinplantsterols,containingrespectively23,17and12mgofplant
sterolsper100gediblefreshweight,whilepassionfruitishighinplantsterols(44mgper100g)[28,31].
However,aswithmostexoticbeverages,thetrueamountofpassionfruitblendedintothedrinkwas
too low to contribute its beneficial effects (<1%). Nevertheless, the concentrations of plant sterols
inthisjuicewereveryhigh,similartotheconcentrationsmeasuredinfreshjuices. Whenhavinga
look at the results for the carrot juice (4.2 Vegetable juices) it can be noted that the concentrations