Determination of Isoflavones in Soybean Flour by Matrix ...downloads.hindawi.com/journals/jfq/2017/8049039.pdf · FS6 74.5±1.6 nd 112.0±2.2 95.4±1.7 39.3±1.1 318.9±8.4 640.5

Research ArticleDetermination of Isoflavones in Soybean Flourby Matrix Solid-Phase Dispersion Extraction and LiquidChromatography with UV-Diode Array Detection

Antonella Aresta1 Pietro Cotugno2 Federica Massari1 and Carlo Zambonin1

1Department of Chemistry University of Bari ldquoAldo Morordquo Via Orabona 4 70125 Bari Italy2CONISMA Piazzale Flaminio 9 00196 Roma Italy

Correspondence should be addressed to Carlo Zambonin carlozamboninunibait

Received 7 March 2017 Accepted 17 July 2017 Published 13 August 2017

Academic Editor Vıtor Spınola

Copyright copy 2017 Antonella Aresta et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A new analyticalmethod based on liquid chromatography (LC)withUV-diode array detection for the simultaneous determinationof daidzein genistein and glycitein and their 7-O-120573-D-glucopyranoside (daidzin genistin and glycitin resp) has been successfullydeveloped All the calibration curves showed good linearity within the concentration range 002ndash2120583gml The limits of detectionand quantitation were 0057 (genistin and glycitein) 0124120583gml (genistein) 0190 120583gml (genistin and glycitein) and 0410 120583gml(genistein) respectively Within-day and between-days precision were found not to be significantly different according to an F-test values ( RSD) ranged from 20 to 29 Extraction and clean-up of soybean flour samples were carried out using matrixsolid-phase dispersion extraction (MSPD) The main parameters affecting extraction yield such as dispersant type and amountof additives cosorbent and extractive solvent were evaluated and optimized The average recovery values were between 857 and1026 The target isoflavone concentration levels estimated in this work fit existing literature data and were comprised between393 and 3453 120583gg The whole procedure has proved to be simple accurate precise and cheap

1 Introduction

Phytoestrogens are a heterogeneous group of naturally occur-ring phenolic compounds with estrogen-like activity [1] Ithas been proposed that plants control male fertility by usingphytoestrogens as part of their natural defense against herbiv-orous animal overpopulation [2 3]

Isoflavones are bioactive metabolites that constitutes awell-known group of phytoestrogens the amounts of whichincrease in the case of plant stress [3 4] In addition theypossess antibiotic and antioxidant properties because of theirability to trap singlet oxygen [5] Epidemiological studieshave suggested a protective effect of isoflavones against anumber of adult chronic diseases including coronary heartdisease as well as breast endometrial and prostate cancers[6ndash8] However their efficacy has not been fully proven sosurveys on risks and benefits are still unclear [9 10]

Isoflavones are present in many legumes in free forms(aglycones) that conjugate with glucose or carbohydrate

moieties [4 11] The unconjugated forms are the bioactivemolecules The ratio between conjugated and unconjugatedforms varies substantially in plants Previous investigationshave shown that genistein and its aglycone are the predom-inant isoflavones in soybean [10 11] Genistein is a specificinhibitor of human DNA topoisomerase II and may possessanticancer activity [12]

Generally the analytical determinations of phytoestro-gens in food have been carried out by LC-UV LC-MS orGC-MS [13ndash20]Their analysis in complexmatrices obviouslyrequires sample pretreatment procedures to simplify thematrix The most used methods for isoflavone determinationare liquid-liquid (LLE) and solid-phase extraction (SPE) [14]Recently solid-phase microextraction (SPME) has also beenproposed for the determination of some major isoflavonesin soy drinks [21] A simple low-cost and relatively quickalternative to the above approaches is matrix solid-phasedispersion (MSPD) It is primarily used because of its flexi-bility and selectivity as well as the possibility of performing

HindawiJournal of Food QualityVolume 2017 Article ID 8049039 5 pageshttpsdoiorg10115520178049039

2 Journal of Food Quality

extraction and clean-up in one step which results in rapidpretreatment and low solvent consumption MSPD involvesdisruption of solid and semisolid samples and simultaneousextraction of the analytes using direct mechanical blendingof the sample with a solid-phase sorbent mainly silica gel oralumina [22] In particular MSPD has been shown to be agood technique for extraction of isoflavonoids in plants usedby alternativemedicine for health care practices [18ndash20] Xiaoet al developed a useful protocol based on MSDP coupledto LC for the determination of daidzein and genistein insoybeans [19] obtaining high extraction efficiencies (greaterthan 80) regardless of the use of lower amounts of sampleand solvents than traditional methods

In this paper MSPD has been successfully used forthe extraction of the isoflavones daidzein genistein andglycitein as well as their glycosides daidzin genistin andglycitin respectively from different defatted soybean floursThemain parameters that affect the extraction of the analytesfrom the matrix such as samplesorbent ratio sorbentsol-vent combination used and volume of elution solvent havebeen carefully evaluated and optimized in order to obtainthe highest possible recoveriesThe instrumental analysis wasperformed by liquid chromatography with UV-diode arraydetection

2 Materials and Methods

21 Materials Daidzein (410158407-dihydroxyisoflavone) genis-tein (4101584057-trihydroxyisoflavone) glycitein (410158407-dihydroxy-6-methoxyisoflavone) and their 7-O-120573-D-glucopyranosides(daidzein genistin and glycitin resp) were from Sigma-Aldrich (Milano Italy) Standards purity was ge95 Stocksolutions of each compound (05mgml) were prepared in amethanoldimethyl sulfoxide (DMSO) mixture (80 20 vv)and stored at minus20∘C in the dark More dilute solutions wereprepared just before use in the solvent A of the mobilephase that is acetonitrilewater (5 95 vv) with 02 formicacid All solvents (Sigma-Aldrich) were LC grade The LCmobile phasewas filtered through a 020120583mnylonmembrane(LabService Analytica Bologna Italy)

22 Apparatus The SPME-LC system consisted of a Spec-tra System Pump model P2000 (ThermoQuest San JoseCA) equipped with a Rheodyne 7125 injection valve (20120583linjection loop) a SCM1000 on-line solvent degasser (ThermoSeparation Products) and a 26 120583mKinetex C18 (10 times 46mmid Phenomenex USA) column The detector was a SpectraSystem model UV6000LP photodiode array (Thermo Finni-gan San Jose CA)

23 Chromatographic and Detection Conditions A binarygradient composed of acetonitrilewater (5 95 vv) with02 formic acid (solvent A) and acetonitrile with 02formic acid (solvent B) was used as mobile phase Thegradient program was 30min linear from 90 A to 40 A1min isocratic at 40 A 1min linear to 90 A 5minequilibration time The flow rate was 07mlmin and thecolumn was kept at room temperature (around 18∘C)

Spectra were acquired in the 240ndash380 nm range at theapex and on the ascending or descending part of each peakPeak purity was checked by spectra overlaying after normal-ization

24 Samples Collection and Pretreatment Defatted soybeanflour samples were purchased from Sigma-Aldrich (SF1) andlocal supermarkets (made in the European Community andintended for human consumption SF2ndashSF6) respectivelySamples were stored at room temperature and kept in adesiccator after opening Spiked samples (5 g each) wereprepared in triplicate by adding suitable amounts of standardsolutions

MSPD was performed as follows 005 g of flour wasmixed with 05 g of anhydrous Na

2SO4 Then 1 g of glass

beads (previously cleaned with ethyl acetate) and 02 g of sil-ica gel sorbent were added to the sample in a mortar andblended for 15min using a pestleThemixturewas transferredinto a syringe barrel equipped with a polypropylene fritcarefully pressed using the syringe plunger and capped witha second frit This step was very important to ensure acalibrated flow of the elution solvent Finally 2ml of ethylacetatewith 2DMSOwas eluted in the syringe and collectedin a vialThen 02ml of the solution was dried under a gentlestream of nitrogen reconstituted in solvent A of the mobilephase and injected into the LC system

25 Statistical Analysis Data from different experimentalgroups of soybean flour samples and concentrations ofisoflavones estimated were compared by a two-way analysesof variance (ANOVA) and differences at 95 level of confi-dence (119901 lt 005) were considered significant

3 Results and Discussion

The first step of the present work was to investigate factorsinfluencing retention and chromatographic efficiency of thetarget compounds in order to optimize their separation LCseparation of phytoestrogens has been generally performed[18] using reverse phase columns and mobile phases consist-ing of methanol (or acetonitrile) and water containing smallamount of acids In the present work a core-shell C18 columnwas adopted and methanol was preferred to acetonitrile aseluent since the latter was the cause of higher column pres-sures Furthermore acidification of the eluent was needed toallow the formation of protonated species formic acid waschosen as amilder alternative to trifluoroacetic or phosphoricacids Eventually the best resolution was achieved using abinary gradient composed of acetonitrilewater with 02formic acid (solvent A) and acetonitrile with 02 formic acid(solvent B) Figure 1 reports an LC-UVDAD chromatogramrelevant to the analysis of the target isoflavones ((1) daidzin(2) glycitin (3) genistin (4) daidzein (5) glycitein and (6)genistein) at the concentration level of 2120583gml using theoptimized mobile phase composition and shows the goodresolution achieved for the target compounds

Calibration curves were then constructed and proved tobe linear in the concentration range 02minus2 120583gml for all theanalytes apart from genistein (04ndash20120583gml) Correlation

Journal of Food Quality 3

1030507090

110130150170190

0 2 4 6 8 10 12 14 16 18 20

Abso

rban

ce (m

AU)

Time (min)

(1)(2)

(3)

(4)

(5)

(6)

minus10

minus30

Figure 1 LC-UVDAD chromatogram obtained by the directinjection of a standard solution of the target isoflavones at theconcentration level of 2120583gml Peak legend (1) daidzin (2) glycitin(3) genistin (4) daidzein (5) glycitein and (6) genistein

Table 1 Estimated LOD and LOQ obtained for the standardisoflavones together with the precision data of the method

Compound LOD(120583gml)

LOQ(120583gml)

RSD()

Daidzin 0070 0234 24Glycitin 0070 0228 27Genistin 0057 0190 26Daidzein 0089 0295 27Glycitein 0057 0190 20Genistein 0124 0410 29

coefficients were better than 0999 and intercepts werenot significantly different from zero at the 95 confidencelevel Each concentration was performed in triplicate Theprecision of the method was investigated at 05 120583gml byperforming replicate measurements (119899 = 5) for 5 days inorder to estimate the within-day and between-days precisionwhich were found not to be significantly different accordingto an F-test and always lower than 3The relevant results arereported in Table 1 The estimated limits of detection (LODs)and limits of quantitation (LOQs) calculated as 3- and 10-fold the standard deviations of the intercept of the calibrationcurves are also listed in Table 1

Experiments were then focused on the optimization ofthe MSPD procedure The selectivity of MSPD depends onmany variables that is samplesorbent ratio sorbentsolventcombination used and volume of elution solvent In thepresent case a ratio of 01 1 2 04 between sample Na

2SO4

glass beads and sorbent (silica gel) respectively was opti-mized to obtain a homogeneous mixture and satisfactoryextraction yields The choice of the nature and the amountof the elution solvent was also crucial since the targetanalytes must be desorbed while the higher number of theremaining matrix components should be retained in thecolumn Different types and volumes of solvents were testedand 2ml of ethyl acetate proved to be the most efficientcombination An increase in analyte solubility in the elution

0

200

400

600

800

0 2 4 6 8 10 12 14 16 18 20

Abso

rban

ce (m

AU)

Time (min)

(1)(2)

(3)

(4)

(5)(6)

Figure 2 LC-UVDAD chromatogram relevant to the analysis of asoybean flour sample extract Peak legend (1) daidzin (2) glycitin(3) genistin (4) daidzein (5) glycitein and (6) genistein

solvent was also achieved by adding 2 of DMSO thusobtaining even higher recoveries

The method was then applied to the determination of thetarget analytes in different soybean flour samples A typicalchromatogram relevant to the analysis of a soybean floursample extract is shown in Figure 2 As can be seen the peaksof the detected analytes were yet again well resolved withno significant interference frommatrix compounds with theonly exception of glycitin which was never detected in anyof the analyzed samples according to literature data [11 12]Each chromatographic peak was confirmed by its retentiontime and the spectra overlaying technique

Recoveries were calculated by using two spiked flours(FS1 and FS2) at different concentration levels and therelevant data are reported in Table 2 High recoveries wereobtained for all the target isoflavones thus confirming thegood extraction efficiency of the developedMSPDprocedureThewithin-day (119899 = 3) coefficients of variation also reportedin Table 2 calculated in the same samples (FS1 and FS2) at thesame concentration levels were always below 6

Quantitation was performed with the standard additionmethod Table 3 lists the isoflavone concentration levelsestimated in the selected soybean flour samples Typicallythe target concentration levels of isoflavones in soybeansand derived products range between 01 and 5mgg [15]Accordingly the levels estimated in this work were between393 and 3453120583gg It is worth noting that the variationof the concentration levels observed between the differentsoybean flours is not statistically significant as confirmedby a two-way ANOVA test Soybean flours can be made indifferent ways in the traditional East Asian way by dry-roasting soybeans or in the modern Western way involvingmoist heating Andrade et al proved that the choice of ovencan modify the content and profile of the different isoflavoneforms in soybean flour [23] Since the analysed sampleswere obtained using the same process the results are notsurprising Furthermore a recent paper reports that signif-icant variances in chemical composition protein profile andisoflavone contents can be found between soybean cultivarsdetermining important differences in their nutritional andfunctional values [24] Our experimental results obtained in


Table 2 Percentage recoveries of isoflavones obtained from two spiked soybean flours (119899 = 3) at different concentration levels together withthe within-day (119899 = 3) coefficients of variation

Sample Isoflavone Spiked concentration(120583gg)

recovery meanplusmn SD

Precision( RSD)

Spiked concentration(120583gg)

recoverymean plusmn SD

Precision( RSD)

FS1

Daidzin 70 882 plusmn 49 56 700 878 plusmn 43 49Glycitin 40 884 plusmn 103 57 400 886 plusmn 39 44Genistin 100 1026 plusmn 61 59 1000 985 plusmn 49 50Daidzein 100 857 plusmn 40 47 1000 862 plusmn 51 59Glycitein 50 875 plusmn 39 45 500 867 plusmn 42 48Genistein 330 950 plusmn 42 44 3300 938 plusmn 39 42

FS2


Table 3 Concentrations of isoflavones estimated (119899 = 3) in the selected soybean flour samples

Sample Daidzin Glycitin Genistin Daidzein Glycitein Genistein Total(120583gg) (120583gg) (120583gg) (120583gg) (120583gg) (120583gg) (120583gg)

FS1 680 plusmn 16 nd 1024 plusmn 21 960 plusmn 17 444 plusmn 11 3200 plusmn 72 6312FS2 690 plusmn 13 nd 1000 plusmn 19 1000 plusmn 19 500 plusmn 12 3300 plusmn 74 6495FS3 667 plusmn 15 nd 920 plusmn 20 980 plusmn 19 448 plusmn 10 3453 plusmn 66 6471FS4 700 plusmn 15 nd 990 plusmn 15 961 plusmn 17 429 plusmn 10 2907 plusmn 75 5990FS5 802 plusmn 17 nd 1104 plusmn 20 897 plusmn 18 500 plusmn 15 3100 plusmn 69 6406FS6 745 plusmn 16 nd 1120 plusmn 22 954 plusmn 17 393 plusmn 11 3189 plusmn 84 6405

selected samples all purchased in Italy indicate that theycould arise from the same cultivar

4 Conclusions

In the agro-food sector fast sensitive and reliable analyticalmethodologies are required in order to allow the matrixcomponents in complex samples such as soybean floursnot only to be highlighted but also to be quantified Thesemethods should also meet logistic requirements that isbeing accurate precise easily transferable and usable alsoin low-technology laboratories for use in the area of man-ufacturing importing or acceptance of raw materials Aselective MSPD-LC-UVDAD analytical method has beensuccessfully developed and validated for the analysis of theisoflavones daidzein genistein and glycitein as well as theirglucosides in soybean flour The results presented in thepaper show that MSPD is a very simple accurate preciseand cheap sample preparation procedure suitable for theisolation of isoflavones from soybean products The targetcompounds were determined in six different soybean floursobtained using the sameprocess and very similar results wereobtained likely indicating a common origin for the samplesAccording to literature data genistein and its glucoside arethe most abundant forms found in analyzed samples

Conflicts of Interest

Theauthors declare there are no conflicts of interest regardingthe publication of this paper

References

[1] C S Hwang H S Kwak H J Lim et al ldquoIsoflavonemetabolitesand their in vitro dual functions They can act as an estrogenicagonist or antagonist depending on the estrogen concentrationrdquoJournal of Steroid Biochemistry and Molecular Biology vol 101no 4-5 pp 246ndash253 2006

[2] C L Hughes Jr ldquoPhytochemical mimicry of reproductive hor-mones andmodulation of herbivore fertility by phytoestrogensrdquoEnvironmental Health Perspectives vol 78 pp 171ndash175 1988

[3] W Barz and R Welle ldquoBiosynthesis and metabolism of isofla-vones and pterocarpan phytoalexins in chickpea soybean andphytopathogenic fungirdquo Phenolic Metabolism in Plants RecentAdvances in Phytochemistry vol 26 pp 139ndash164 1992

[4] R A Dixon ldquoNatural products and plant disease resistancerdquoNature vol 411 no 6839 pp 843ndash847 2001

[5] K B Pandey and S I Rizvi ldquoPlant polyphenols as dietary anti-oxidants in human health and diseaserdquo Oxidative Medicine andCellular Longevity vol 2 no 5 pp 270ndash278 2009

[6] C D Haines P A Harvey E D Luczak et al ldquoEstrogenic com-pounds are not always cardioprotective and can be lethal in


males with genetic heart diseaserdquo Endocrinology vol 153 no9 pp 4470ndash4479 2012

[7] E Zhao and QMu ldquoPhytoestrogen biological actions onmam-malian reproductive system and cancer growthrdquo Scientia Phar-maceutica vol 79 no 1 pp 1ndash20 2011

[8] L Vitetta A Sali and S Coulson ldquoSoy and health The latestevidencerdquoMedicine Today vol 12 no 3 pp 67ndash74 2011

[9] S Andres K Abraham K E Appel and A Lampen ldquoRisks andbenefits of dietary isoflavones for cancerrdquo Critical Reviews inToxicology vol 41 no 6 pp 463ndash506 2011

[10] M Messina and V L Messina ldquoExploring the soyfood contro-versyrdquo Nutrition Today vol 48 no 2 pp 68ndash75 2013

[11] S Barnes ldquoThe biochemistry chemistry and physiology of theisoflavones in soybeans and their food productsrdquo LymphaticResearch and Biology vol 8 no 1 pp 89ndash98 2010

[12] Y Mizushina K Shiomi I Kuriyama Y Takahashi and HYoshida ldquoInhibitory effects of a major soy isoflavone genisteinon human DNA topoisomerase II activity and cancer cell pro-liferationrdquo International Journal of Oncology vol 43 no 4 pp1117ndash1124 2013

[13] QWuMWang and J E Simon ldquoAnalytical methods to deter-mine phytoestrogenic compoundsrdquo Journal of ChromatographyB Analytical Technologies in the Biomedical and Life Sciencesvol 812 no 1-2 pp 325ndash355 2004

[14] M A Rostagno A Villares E Guillamon A Garcıa-Lafuenteand J A Martınez ldquoSample preparation for the analysis ofisoflavones from soybeans and soy foodsrdquo Journal of Chro-matography A vol 1216 no 1 pp 2ndash29 2009

[15] S Barnes M Kirk and L Coward ldquoIsoflavones and theirconjugates in soy foods Extraction conditions and analysis byHPLC-mass spectrometryrdquo Journal of Agricultural and FoodChemistry vol 42 no 11 pp 2466ndash2474 1994

[16] A Zafra-Gomez A Garballo L E Garcıa-Ayuso and J CMorales ldquoImproved sample treatment and chromatographicmethod for the determination of isoflavones in supplementedfoodsrdquo Food Chemistry vol 123 no 3 pp 872ndash877 2010

[17] G Fiechter B Raba A Jungmayr and H K Mayer ldquoCharac-terization of isoflavone composition in soy-based nutritionalsupplements via ultra performance liquid chromatographyrdquoAnalytica Chimica Acta vol 672 no 1-2 pp 72ndash78 2010

[18] B Barfi M R Hadjmohammadi and M R Kasaai ldquoDetermi-nation of daidzein and genistein in soybean and its waste bymatrix solid-phase dispersion extraction and HPLCrdquo Monat-shefte fur Chemie vol 140 no 10 pp 1143ndash1148 2009

[19] H B Xiao M Krucker K Albert and X M Liang ldquoDeter-mination and identification of isoflavonoids in Radix astra-gali by matrix solid-phase dispersion extraction and high-performance liquid chromatographywith photodiode array andmass spectrometric detectionrdquo Journal of Chromatography Avol 1032 no 1-2 pp 117ndash124 2004

[20] E de Rijke F de Kanter F Ariese U A T Brinkman and CGooijer ldquoLiquid chromatography coupled to nuclear magneticresonance spectroscopy for the identification of isoflavone glu-coside malonates in T pratense L leavesrdquo Journal of SeparationScience vol 27 no 13 pp 1061ndash1070 2004

[21] A Aresta F Di Grumo and C Zambonin ldquoDetermination ofMajor Isoflavones in Soy Drinks by Solid-Phase Micro Extrac-tion Coupled to Liquid Chromatographyrdquo Food AnalyticalMethods vol 9 no 4 pp 925ndash933 2016

[22] S A Barker ldquoMatrix solid phase dispersion (MSPD)rdquo Journal ofBiochemical and Biophysical Methods vol 70 no 2 pp 151ndash1622007

[23] J C Andrade J M GMandarino L E Kurozawa and E I IdaldquoThe effect of thermal treatment of whole soybean flour on theconversion of isoflavones and inactivation of trypsin inhibitorsrdquoFood Chemistry vol 194 pp 1095ndash1101 2016

[24] S Ciabotti A C B B Silva A C P Juhasz et al ldquoChemicalcomposition protein profile and isoflavones content in soybeangenotypes with different seed coat colorsrdquo International FoodResearch Journal vol 23 no 2 pp 621ndash629 2016

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


extraction and clean-up in one step which results in rapidpretreatment and low solvent consumption MSPD involvesdisruption of solid and semisolid samples and simultaneousextraction of the analytes using direct mechanical blendingof the sample with a solid-phase sorbent mainly silica gel oralumina [22] In particular MSPD has been shown to be agood technique for extraction of isoflavonoids in plants usedby alternativemedicine for health care practices [18ndash20] Xiaoet al developed a useful protocol based on MSDP coupledto LC for the determination of daidzein and genistein insoybeans [19] obtaining high extraction efficiencies (greaterthan 80) regardless of the use of lower amounts of sampleand solvents than traditional methods

In this paper MSPD has been successfully used forthe extraction of the isoflavones daidzein genistein andglycitein as well as their glycosides daidzin genistin andglycitin respectively from different defatted soybean floursThemain parameters that affect the extraction of the analytesfrom the matrix such as samplesorbent ratio sorbentsol-vent combination used and volume of elution solvent havebeen carefully evaluated and optimized in order to obtainthe highest possible recoveriesThe instrumental analysis wasperformed by liquid chromatography with UV-diode arraydetection

2 Materials and Methods

21 Materials Daidzein (410158407-dihydroxyisoflavone) genis-tein (4101584057-trihydroxyisoflavone) glycitein (410158407-dihydroxy-6-methoxyisoflavone) and their 7-O-120573-D-glucopyranosides(daidzein genistin and glycitin resp) were from Sigma-Aldrich (Milano Italy) Standards purity was ge95 Stocksolutions of each compound (05mgml) were prepared in amethanoldimethyl sulfoxide (DMSO) mixture (80 20 vv)and stored at minus20∘C in the dark More dilute solutions wereprepared just before use in the solvent A of the mobilephase that is acetonitrilewater (5 95 vv) with 02 formicacid All solvents (Sigma-Aldrich) were LC grade The LCmobile phasewas filtered through a 020120583mnylonmembrane(LabService Analytica Bologna Italy)

22 Apparatus The SPME-LC system consisted of a Spec-tra System Pump model P2000 (ThermoQuest San JoseCA) equipped with a Rheodyne 7125 injection valve (20120583linjection loop) a SCM1000 on-line solvent degasser (ThermoSeparation Products) and a 26 120583mKinetex C18 (10 times 46mmid Phenomenex USA) column The detector was a SpectraSystem model UV6000LP photodiode array (Thermo Finni-gan San Jose CA)

23 Chromatographic and Detection Conditions A binarygradient composed of acetonitrilewater (5 95 vv) with02 formic acid (solvent A) and acetonitrile with 02formic acid (solvent B) was used as mobile phase Thegradient program was 30min linear from 90 A to 40 A1min isocratic at 40 A 1min linear to 90 A 5minequilibration time The flow rate was 07mlmin and thecolumn was kept at room temperature (around 18∘C)

Spectra were acquired in the 240ndash380 nm range at theapex and on the ascending or descending part of each peakPeak purity was checked by spectra overlaying after normal-ization

24 Samples Collection and Pretreatment Defatted soybeanflour samples were purchased from Sigma-Aldrich (SF1) andlocal supermarkets (made in the European Community andintended for human consumption SF2ndashSF6) respectivelySamples were stored at room temperature and kept in adesiccator after opening Spiked samples (5 g each) wereprepared in triplicate by adding suitable amounts of standardsolutions

MSPD was performed as follows 005 g of flour wasmixed with 05 g of anhydrous Na

2SO4 Then 1 g of glass

beads (previously cleaned with ethyl acetate) and 02 g of sil-ica gel sorbent were added to the sample in a mortar andblended for 15min using a pestleThemixturewas transferredinto a syringe barrel equipped with a polypropylene fritcarefully pressed using the syringe plunger and capped witha second frit This step was very important to ensure acalibrated flow of the elution solvent Finally 2ml of ethylacetatewith 2DMSOwas eluted in the syringe and collectedin a vialThen 02ml of the solution was dried under a gentlestream of nitrogen reconstituted in solvent A of the mobilephase and injected into the LC system

25 Statistical Analysis Data from different experimentalgroups of soybean flour samples and concentrations ofisoflavones estimated were compared by a two-way analysesof variance (ANOVA) and differences at 95 level of confi-dence (119901 lt 005) were considered significant

3 Results and Discussion

The first step of the present work was to investigate factorsinfluencing retention and chromatographic efficiency of thetarget compounds in order to optimize their separation LCseparation of phytoestrogens has been generally performed[18] using reverse phase columns and mobile phases consist-ing of methanol (or acetonitrile) and water containing smallamount of acids In the present work a core-shell C18 columnwas adopted and methanol was preferred to acetonitrile aseluent since the latter was the cause of higher column pres-sures Furthermore acidification of the eluent was needed toallow the formation of protonated species formic acid waschosen as amilder alternative to trifluoroacetic or phosphoricacids Eventually the best resolution was achieved using abinary gradient composed of acetonitrilewater with 02formic acid (solvent A) and acetonitrile with 02 formic acid(solvent B) Figure 1 reports an LC-UVDAD chromatogramrelevant to the analysis of the target isoflavones ((1) daidzin(2) glycitin (3) genistin (4) daidzein (5) glycitein and (6)genistein) at the concentration level of 2120583gml using theoptimized mobile phase composition and shows the goodresolution achieved for the target compounds

Calibration curves were then constructed and proved tobe linear in the concentration range 02minus2 120583gml for all theanalytes apart from genistein (04ndash20120583gml) Correlation


1030507090

110130150170190

0 2 4 6 8 10 12 14 16 18 20

Abso

rban

ce (m

AU)

Time (min)

(1)(2)

(3)

(4)

(5)

(6)

minus10

minus30




LOQ(120583gml)

RSD()




2SO4


0

200

400

600

800

0 2 4 6 8 10 12 14 16 18 20

Abso

rban

ce (m

AU)

Time (min)

(1)(2)

(3)

(4)

(5)(6)










Precision( RSD)



Precision( RSD)

FS1


FS2






4 Conclusions




References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1030507090

110130150170190

0 2 4 6 8 10 12 14 16 18 20

Abso

rban

ce (m

AU)

Time (min)

(1)(2)

(3)

(4)

(5)

(6)

minus10

minus30




LOQ(120583gml)

RSD()




2SO4


0

200

400

600

800

0 2 4 6 8 10 12 14 16 18 20

Abso

rban

ce (m

AU)

Time (min)

(1)(2)

(3)

(4)

(5)(6)










Precision( RSD)



Precision( RSD)

FS1


FS2






4 Conclusions




References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Precision( RSD)



Precision( RSD)

FS1


FS2






4 Conclusions




References


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Determination of Isoflavones in Soybean Flour by Matrix ...downloads.hindawi.com/journals/jfq/2017/8049039.pdf · FS6 74.5±1.6 nd 112.0±2.2 95.4±1.7 39.3±1.1 318.9±8.4 640.5