Validation of an HPLC-DAD method for the determination of plant phenolics

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w ww . e l s e v i e r . c o m / l o c a t e / b j p

Short communication

Validation of an HPLC-DAD method for the determination of plant phenolics

Rosa M. González-González

^a,1

, Lina Barragán-Mendoza

^a,1

, Ana L. Peraza-Campos

^a

, Roberto Mu ˜ niz-Valencia

^a

, Silvia G. Ceballos-Maga ˜ na

^b,∗

, Hortensia Parra-Delgado

^a,∗

aFacultaddeCienciasQuímicas,UniversidaddeColima,Coquimatlán,Colima,México

bFacultaddeCiencias,UniversidaddeColima,Colima,México

a r t i c l e i n f o

Articlehistory:

Received11June2019 Accepted14June2019

Keywords:

Phenolics Aqueousextracts Analysis Medicinalplants

a b s t r a c t

Aselective,sensitiveandprecisereversedphaseHPLC-DADmethodwasdevelopedandvalidatedforthe simultaneousdeterminationofsixphenolicacidsintheaqueousextractandtheirhydrolyzedformspre- paredfromSolanumelaeagnifoliumCav.,Solanaceae,Ampelocissusacapulcensis(Kunth)Planch.,Vitaceae, orBrosimumalicastrumSw.,Moraceae.Thenewmethodshowedgoodlinearity(r>0.999)inarelatively wideconcentrationrange(0.5–100mg/l).Thelimitsofdetectionandquantiﬁcationforthecompounds wereintherangeof0.097–0.467mg/land0.097–0.496mg/l,respectively.Therecoveriesofcompounds werecalculatedinthreedifferentconcentrationsintherangeof88.07–109.17%andmatrixeffectwas lessthan5%forallphenolicacids.Finally,ourdevelopedHPLCmethodissimple,reliableandsuccessfully appliedtoidentifyandquantifythephenolicacidsincomplexaqueousextractsfrommedicinalspecies, thatcanbeusefulfortheanalysisofinfusionsthatpeopleconsumeinfolkmedicine.

Introduction

Medicinalplantshavebeenusedinthetreatmentofdiseases that affectthepopulation. However,the peopleconsumethem despitetheknowledgeabouttheirchemicalcompositionisrather limited(Piresetal.,2017).

Nowadays, there is a growing interest in the analysis and identificationof medicinal plants constituents,mainly phenolic compounds (Ibrahim et al., 2015).Such secondary metabolites comprisealargevarietyofcompounds:simpleflavonoids,tannins, lignin,complexflavonoids,anthocyanins,andphenolicacids(Lin etal.,2016).Theselatestoccurindifferentformssuchas,aglycones, esters,glycosides,and/orboundcomplexes(Rossetal.,2009)and theypossessabroadspectrumofpharmacologicalactionssuchas antibacterialandantifungalactivity,anticarcinogenic,cardiopro- tective,antiviral,andantiallergic(Wenetal.,2005;Abad-García etal.,2007).

AspartofourstudiesofMexicanmedicinalplants,weincluded theanalysisof phenoliccompounds ofthree medicinalspecies,

∗ Correspondingauthors.

E-mails:silvia[email protected](S.G.Ceballos-Maga ˜na),[email protected](H.

Parra-Delgado).

1Bothauthorscontributedequallytothiswork.

SolanumelaeagnifoliumCav.,Solanaceae,Ampelocissusacapulcensis (Kunth)Planch.,Vitaceae,andBrosimumalicastrum.Sw.,Moraceae.

Brosimumalicastrum(knownasmojoorRamon)isatreeconsid- eredasmultipurposespecies,becauseallitspartscanbeemployed indifferenttypesofapplications.Theseeds,leaves,latex,andbark of mojoare medicinally usedin various regions of Mexicoand Guatemala.Anextractofthecrushedseedsisrecommendedas agalactogenwhichstimulatesthemilkproduction.Leafinfusions areemployedascoughsuppressantsandinthetreatmentofkid- neyailments.Atonicmadefromthebarkisusedtotreatchest pains,asthmaandcancer(Ortizetal.,1995).S.elaeagnifolium(com- monnametomatillo)isaperennialshrubofSolanaceaefamily.This planthastraditionallybeenusedforthetreatmentofcancer,sore throats,toothaches,and gastrointestinaldisorders(Houdaetal., 2014).Finally,A.acapulcensis(commonnameuvacimarrona)isa scandentshrubnativefromMexicothathasbeenusedintradi- tionalmedicineforthetreatmentofdisordersofthegenitourinary system(Vergara-Santanaetal.,2013).

Theextractionofphenolicacidsiscarriedoutwithaqueous- organic solvents to obtain soluble polyphenols followed by a hydrolysistreatmenttoobtainfreepolyphenols,wheretheacidand basichydrolysisarethemostcommon(ArranzandSaura-Calixto, 2010;Ozer,2017).SeveralHPLCmethodshavebeenreportedto quantifythese compoundsinextractsfrom plants(Sinhaetal., 2007;Farzaeietal.,2014;Ozer,2017;Piresetal.,2017).However,

https://doi.org/10.1016/j.bjp.2019.06.002

creativecommons.org/licenses/by-nc-nd/4.0/).

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existingmethodsareusedfororganicextractsandtherearefew reportsforthedeterminationofphenolicacidsinaqueousextracts (DeSouzaetal.,2002;ZiakováandBrandˇsteterová,2003).Taking intoaccountthatseveralremediesarepreparedasinfusions,itis stillimportantthedevelopmentofmethodsforaqueousmatrices.

Therefore,inthispaper,wereportanewHPLC-DADmethodforthe determinationofphenolicacidsthatwasvalidatedandappliedto theanalysisofaqueousextractsofdifferentmedicinalplants.

Materialsandmethods

HPLC-grademethanol(MeOH)andacetonitrile(ACN)werepur- chasedfromJ.T.Baker-company(Guadalajara,Mexico).Ultrapure- water (Milli-Q)with18.2M/cm resistivitywasobtainedfrom MillipakExpress FilterUnitequipment(Millipore, Bedford,MA, USA). Standards of gallic (GA), vanillic (VA), p-hydroxybenzoic (p-HBA), caffeic (CA), p-coumaric (p-CoA), and trans-cinnamic (trans-CA)acidswerepurchasedfromSigma–Aldrich(St.Louis,MO, USA).

Fruits,leaves and bark of B. alicastrum were collectedfrom Suchitlan,Colima,Mexico(19^◦2355.0N103^◦4521.9W)inMarch 2015. Leaves of S. elaeagnifolium were obtained from Alpuye- quito,Colima,Mexico(19^◦1026.1N103^◦4318.3W)inSeptember 2014.RootofA.acapulcensiswerecollectedfromTecoman,Colima, Mexico(18^◦5300.6N103^◦4353.8W)inMay2013.Thevegetal materialofthree specieswasdriedin convectionovenat37^◦C for12h.Thesamplesweremilledusingamanualmill.Theﬂour obtainedwasstoredinaglasshermeticrecipientandstoredindark at4^◦C.

Botanicalidentiﬁcationofthreespecieswaskindlyprovidedby M.S.RafaelTorres-ColinofNationalHerbariumofMexico(MEXU), InstituteofBiology,UNAM,Mexico.Voucherherbariumspecimens forAmpelocissusacapulcensis(Kunth)Planch.,Vitaceae,(1453310), SolanumelaeagnifoliumCav.,Solanaceae(1423235),Brosimumali- castrumSw.,Moraceae(1457305),weredepositedatMEXU.

HPLC-DADanalyseswereperformedonanAlliancee2695sep- arationmodule,consistingofaquaternarypump,onlinevacuum degassing,autosampler,andcolumnovencoupledtoa2996DAD withawavelengthrangeof190–800nmfromWaters(Milford,MA, USA).DatacollectionandanalysiswereperformedusingEmpower Pro2 SoftwarefromWaters (Milford,MA,USA). For chromato- graphic separation, first, two columns were used, XBridge-C18 column(4.6mm×150mm,3.5␮m) fromWaters(Wexford,Ire- land)andAscentis-ExpressRP-Amidecolumn(4.6mm×250mm, 5␮m) from Sigma–Aldrich (St. Louis, MO, USA). For the chro- matographicseparations of phenolic compounds, the following conditions were employed, an Ascentis Express RP-Amide col- umnata0.7ml/minflow-rate,25^◦C,andaninjectionvolumeof 15␮l.AlineargradientprofileofmobilephaseA(0.1%V/Vformic acid/ultrapure-water)and mobilephase B(methanol),thatcon- sistedin15–80%Bover60min,15%Bat70min.Totalruntimewas 70min.Identificationofthephenoliccompoundswasachievedby comparingretentiontimesandUVspectraoftheunknownswith thestandards.

Consideringtheethnomedicalinformationof eachplant,dif- ferent aqueous extracts were prepared. Complete information aboutstandardsolutionsandpreparationofthesamples,andtheir hydrolysis(Rossetal.,2009)canbefoundinSupplementarydata.

Validationoftheproposedmethodwascarriedoutaccordingto theInternationalConferenceonHarmonization(ICH,1996/2005) and the Commission Decision (2002/657/EC) guidelines. The methodwasvalidatedthroughestimationofselectivity,linearity, precision,accuracy,andsensibility(Supplementarydata).

ThematrixeffectwasassessedaccordingwiththeEuropean Commission(SANTE/11945/2015)bycomparingtheslopeofcali-

brationcurveofstandardsandthecurveobtainedaftertheaddition of thedifferent concentrations of thestandards to thesample.

Thematrixeffectwasdeterminedasfollows(Gómez-Ramosetal., 2016):

Matrixeffect (%) =

slope_standard slope_spiked

−1

×100

Resultsanddiscussion

Some HPLC methods reported for the analysis of phenolic compounds, in medicinal plants, useorganic solvents for their extraction,e.g.methanol/acetone,ethanol/water,methanol/water, ethylacetate,butanoletc.,(Ozer,2017;Piresetal.,2017).However, mostpeopleuseherbalremediesintheformofinfusionortea(Pires etal.,2017).Forthisreason,inthepresentstudyweproposeda HPLCmethodwhichcanbeusedforaqueousextractsandapplied fortheanalysisofthreemedicinalplantsoftheregion,inorderto identifyandquantifythephenoliccompoundsconsumedbyaper- sonwhendrinkingtheinfusion.Consideringthatsomephenolic acidsarenotfree,itisnecessarytohydrolyzethem.Therefore,the extractsweresequentiallyhydrolyzed,ﬁrstusingmethanol/acetic acid, then10MNaOHand ﬁnallyconcentratedHCl (Rossetal., 2009).

Fortheoptimizationofthemethodseveralconditions,solvents, timesandcolumntypeswereusedtoobtaintheoptimumsepa- rationofeachstandardintheextracts.Twomobilephaseswere tested, MeOH:H₂O/formic acid (0.1%) and ACN:H₂O/phosphoric acid(0.1%). Both mobilephases allowedthe correct separation ofthestandards,however,MeOH:H2O/formicacid(0.1%)wasthe phase with better resolution and separation of phenolic com- poundswithintheaqueousextracts.Besides,variousﬂow rates (0.5,0.7,1and1.2ml/min)andcolumntypes(XBridge-C18column andAscentis-RPamidecolumn)wereemployed.XBridge-C18pre- sentedanoverlapofphenolicacids.UsingtheAscentis-RPamide, allthephenolicacidswerebaselineseparatedwithatotalanalysis timeof70min.Chromatogramswereacquiredatdifferentwave- lengthsaccordingtoabsorptionmaximaofanalyzedcompounds.

Thesewere270,292,260,324,310,and277nmforGA,VA,p-HBA, CA,p-CoA,andtrans-CA,respectively.

TheestablishedHPLCmethodwasvalidatedintermsofselec- tivity, linearity, precision, accuracy, and sensibility according to ICH and Commission Decision guidelines (ICH, 1996/2005;

2002/657/EC).ThevalidationparametersareshowninTable1.

Method selectivity was evaluated by overlapping the chro- matogramofthestandardsolutionwiththatofthesamples.Also, thecomparisonoftheUVspectrawascarriedout(Fig.1).

Linearitywasevaluatedbyusingstandardsolutionsdissolved in 15:85 mobile phase B/A at concentrations in the range of 5–75mg/lforGAandp-HBA,1–75mg/lforVA,0.5–100mg/lfor CA,1–100mg/lforp-CoA,and2.5–50mg/lfortrans-CA.Eachcon- centrationwasanalyzedintriplicate.Thecalibrationcurveswere constructedfrompeakareasofthereferencecompoundsversus theirconcentrations(Fig.1A, Supplementarydata).Thecorrela- tioncoefﬁcients of all thecalibrationcurves weregreater than 0.999.Theprecisionofthemethodwasdeterminedasrepeata- bility and reproducibilityin terms of percentrelative standard deviation(%RSD).The%RSDvalues forevaluatedconcentrations (10,25and50mg/l)werelowerthan2.18%.Recoverywasdeter- minedemployingtheextractsinwhichthepeakswereclearand withoutinterferences. Therefore, for GA theprocess wasmade intheleavestotalextractofB.alicastrum;meanwhileforVA,p- HBA, CA,p-CoA, and trans-CA acids the procedurewas carried outinthe“basichydrolysis”ofB.alicastrum.Theresultsobtained show percentages of recovery between 88.07-109.17. These

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Table1 ValidationparametersfortheHPLC–DADmethodusedforthedeterminationofphenolicacidsindifferentextractsofmedicinalplants. Precision(RSDg%)Accuracy AnalytetRa(min)Linearrange(mg/l)rbmcbdLODe(mg/l)LOQf(mg/l)RepeatabilityReproducibilityRecovery(%)Matrixeffect(%) C1hC2iC3jC1hC2iC3jC1hC2iC3j GA11.865–750.999831616−241090.46650.49581.170.652.181.130.311.29106.4091.1895.22−3.18 VA27.191–750.999760045−8584.20.11810.12440.470.461.140.680.300.09103.5897.1897.791.26 p-HBA28.895-750.999660696−214110.41610.47670.400.290.170.530.210.0899.9688.0797.94−4.75 CA30.000.5–1000.9996243407−179620.09650.09670.571.051.490.410.210.27108.31103.03102.070.55 p-CoA37.971–1000.999593693−273830.27580.29040.390.810.150.550.820.66109.17101.23102.312.13 trans-CiA45.492.5–500.9995159044−2034140.17210.18160.210.290.050.410.110.0496.88102.49101.121.96 GA,gallicacid;VA,vanillicacid;p-HBA,p-hydroxybenzoicacid;CA,caffeicacid;p-CoA,p-coumaricacid;trans-CA,trans-cinnamicacid. aRetentiontime(min). bCorrelationcoefﬁcient. cSlope. dIntercept. eLimitofdetection. fLimitofquantiﬁcation. gRelativestandarddeviation. hLowconcentrationlevelof10mg/l. iMiddleconcentrationlevelof25mg/l. jHighconcentrationlevelof50mg/l.

Fig. 1.HPLC chromatogramsobtained for the standard phenolic compounds employedintheoptimizationofchromatographicmethodat270nm(A).Totalaque- ousextract(B)andbasichydrolysis(C)ofleavesBrosimumalicastrumSwartz.GA:

gallicacid;VA:vanillicacid;p-HBA:p-hydroxybenzoicacid;CA:caffeicacid;p-CoA:

p-coumaricacid;trans-CA:trans-cinnamicacid.

precisionandrecoveryvaluesareconsideredacceptableaccording toCommissionDecision(2002/657/EC).

LODvalueswerecalculatedbetween0.097and0.467mg/land theLOQvalueswerefoundintherangeof0.097–0.496mg/l,indi- catinghighsensibilityofthemethod.

Matrix effect(ME) isconsideredthe responseof ananalyte, eitherpositiveornegative,causedbycoelutingcompounds,rel- ativetoaninjectionofapurestandard,anditspresenceisrelated tothesamplenature,affectingreproducibility,linearity,andaccu- racyofthemethods(Cappielloetal.,2010).Becauseofextracts areusuallyconstitutedbycomplexmixtureofcompounds,inthis work we considered important to include a ME study. It was explored by comparingthe external calibration slope with the matrixmatchcalibrationslope.Theresultsobtainedshowedsignal suppressionforGAandp-HBA,whiletherewasasignalenhance- mentforVA,CA,p-CoAandtrans-CA.Inallcases,itwaslessthan

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Table2

ContentofphenolicacidsinaqueousextractsofBrosimumalicastrum(leaves,barkandseeds),Solanumelaeagnifolium(leaves)andAmpelocissusacapulcensis(root)andtheir hydrolysis.

GA(␮g/mgof lyophilizate extract)

VA(␮g/mgof lyophilizate extract)

p-HBA(␮g/mgof lyophilizate extract)

CA(␮g/mgof lyophilizate extract)

p-CoA(␮g/mgof lyophilizate extract)

trans-CiA(␮g/mg oflyophilizate extract) B.alicastrumleaves

Totalextract 2.34±0.007 n/d n/d n/d n/d n/d

MeOH/AcAhydrolysis 0.91±0.081 n/d n/d 0.15±0.038 n/d n/d

Basichydrolysis n/d 1.44±0.010 n/d 0.71±0.0048 n/d n/d

B.alicastrumbark

Totalextract n/d n/d n/d n/d n/d n/d

MeOH/AcAhydrolysis n/d n/d n/d n/d n/d n/d

Basichydrolysis n/d 19.44±0.253 11.55±0.054 n/d n/d n/d

Acidhydrolysis n/d 0.58±0.022 0.30±0.002 0.06±0.001 n/d n/d

B.alicastrumseed

Totalextract n/d 0.65±0.005 n/d n/d n/d n/d

MeOH/AcAhydrolysis n/d n/d n/d n/d n/d n/d

Basichydrolysis n/d 0.10±0.014 n/d 0.0058±0.002 0.013±0.0004 n/d

Acidhydrolysis n/d 0.09±0.012 n/d n/d n/d n/d

S.elaeagnifoliumleaves

Totalextract n/d n/d n/d n/d n/d n/d

MeOH/AcAhydrolysis n/d n/d n/d 0.04±0.033 0.01±0.006 n/d

Basichydrolysis n/d 0.35±0.015 0.77±0.015 0.11±0.003 0.89±0.014 n/d

Acidhydrolysis n/d 0.22±0.025 0.32±0.009 0.10±0.0003 n/d n/d

A.acapulcensisroot

Totalextract 6.01±0.023 n/d n/d n/d n/d n/d

MeOH/AcAhydrolysis 1.47±0.180 n/d n/d n/d n/d n/d

Basichydrolysis 5.34±0.052 n/d 1.57±0.008 n/d 3.35±0.057 n/d

Acidhydrolysis n/d n/d 0.51±0.006 n/d 1.45±0.013 n/d

GA,gallicacid;VA,vanillicacid;p-HBA,p-hydroxybenzoicacid;CA,caffeicacid;p-CoA,p-coumaricacid,trans-CA:trans-cinnamicacid,n/d,notdetected.

5%whichcanbeacceptedaccordingwithEuropeanCommission (SANTE/11945/2015).

In order to demonstrate the applicability of the developed method,threedifferentmedicinalplantswereanalyzed.Thecon- tentofphenolicacidsinaqueoustotalextractsandtheirhydrolyzed formsisshowninTable2.Phenoliccompoundsthatwerefound freeinthetotalaqueousextractsweretheGA(2.34␮g/mgLE)in theleavesofB.alicastrum(Fig.1)andintherootofA.acapulcensis (6.01␮g/mgLE)(Fig.4A–E1,Supplementarydata),whilevanillic acid(0.65␮g/mgLE)wasfoundintheseedsofB.alicastrum(Fig- ure3A–C1,Supplementarydata).Furthermore,thenewmethod allowedthedeterminationandquantiﬁcationofthephenolicsin thehydrolyzed. Our resultsindicated thatmostof thephenolic acidsintheextractsarejoinedtoothercompoundsforminginsol- ubleboundcomplexes(Rossetal.,2009).

Conclusion

TheHPLCmethodfortheanalysisofsixphenolicacidsintotal aqueousextractsofB.alicastrum,S.elaeagnifoliumandA.acapul- censisandtheirhydrolyzedformswasdevelopedandvalidated.

Themethodshowedgoodlinearity, precision,sensibility,selectivity,andrecovery.Inaddition,thematrixeffectwas<5%forall phenolicacids.Thus,thedevelopedmethodissimple,reliableand successfullyappliedtoidentifyandquantifyphenolicacidsincom- plexaqueousextractsfromdiversepartsofaplantanddifferent medicinalspecies.

Authors’contributions

LBMandRMGGcontributionincludedHPLCanalysis,analyzing theresultsandpreparingthepaper.SGCM,RMV,ALPCandHPD contributionincludedanalyzingresultsandpreparingthepaper.

Allauthorshavereadtheﬁnalmanuscriptandapprovedthesub- mission.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

Ethicaldisclosures Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Conﬁdentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Acknowledgements

LBMandRMGGthankstoCONACYT-MEXICOforthescholarship (Reg. Number 631256 and 633587, respectively). Acknowledg- mentstoMiguel-A.Maldonado-Michel,FernandoJiménez-Beltrán, Miriam-R.Rodríguez-Alejandrez,andMario-A.Gaitán-Hinojosafor theirhelpinthecollectionofvegetalsamplesandpreparationofthe lyophilizedextracts.FinancialsupportfromPRODEPisacknowl- edged.

AppendixA. Supplementarydata

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/

j.bjp.2019.06.002.

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