Journal of Chromatography A, 1216 (2009) 2–29

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Journal of Chromatography A

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Review

Sample preparation for the analysis of isoflavones from soybeans and soy foods

M.A. Rostagnoa,∗, A. Villaresa, E. Guillamóna, A. García-Lafuentea, J.A. Martíneza,b

a Centro para la Calidad de los Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA),Campus Universitario “Duques de Soria”, 42004 Soria, Spainb Universidad de Navarra, Dpto. Fisiología y Nutrición, Edificio de Investigación, C/Irunlarrea, 1, 31008 Pamplona, Spain

a r t i c l e i n f o

Article history:Received 6 August 2008Received in revised form 3 November 2008Accepted 13 November 2008Available online 19 November 2008

Keywords:ReviewsIsoflavonesSoybeans

a b s t r a c t

This manuscript provides a review of the actual state and the most recent advances as well as currenttrends and future prospects in sample preparation and analysis for the quantification of isoflavonesfrom soybeans and soy foods. Individual steps of the procedures used in sample preparation, includ-ing sample conservation, extraction techniques and methods, and post-extraction treatment proceduresare discussed. The most commonly used methods for extraction of isoflavones with both conventionaland “modern” techniques are examined in detail. These modern techniques include ultrasound-assistedextraction, pressurized liquid extraction, supercritical fluid extraction and microwave-assisted extraction.Other aspects such as stability during extraction and analysis by high performance liquid chromatographyare also covered.

Sample conservationSample preparation

© 2008 Elsevier B.V. All rights reserved.

ExtractionAnalysis

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32. General aspects of soy isoflavones determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43. Sample stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44. Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65. Extraction techniques and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

5.1. Solid and semi-solid samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75.1.1. Conventional extraction methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75.1.2. Modern extraction techniques and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

5.2. Liquid samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215.3. Optimization of extraction conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225.4. Critical comparison of extraction methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

6. Post-treatment of extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

7. Separation approaches/techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Abbreviations: ε, Dielectric constant; ACE, Acetone; ADi, Acetyl daidzin; AGi, Acetyl genistin; AGly, Acetyl glycitin; ASE, Accelerated solvent extraction; CE, Capillaryelectromigration techniques; De, Daidzein; Di, Daidzin; DMSO, Dimethylsulfoxide; DSM, Defatted soybean meal; EtOH, Ethanol; Ge, Genistein; Gi, Genistin; Gle, Glycitein; Gly,Glycitin; MAE, Microwave-assisted extraction; MeCN, Acetonitrile; MeOH, Methanol; MGi, Malonyl genistin; MDi, Malonyl Daidzin; MGly, Malonyl glycitin; PLE, Pressurizedliquid extraction; PSE, Pressurized solvent extraction; SC-CO2, Supercritical CO2; SFE, Supercritical fluid extraction; SPE, Solid phase extraction; SPI, Soy protein isolate; SPME,Solid phase microextraction; SWE, Superheated water extraction; UAE, Ultrasound-assisted extraction.

∗ Corresponding author. Tel.: +34 975 233204; fax: +34 975 233205.E-mail address: rostagno.mauricio@inia.es (M.A. Rostagno).

0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.chroma.2008.11.035

romatogr. A 1216 (2009) 2–29 3

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. Introduction

Functional foods are one of the most promising fields concerningutritional sciences. These food-stuffs are interesting from the con-umer point of view with the prospect of maintaining health andreventing diseases by using natural foods as part of the habitualiet, and also from the industry point of view, for the added valuef the products. There are several raw materials that can be used forealthy purposes and soybeans are among those with the greatestotential. Soybeans are one of most produced and commercializedommodities worldwide. Actually, there are several foods derivedr based on soybeans such as soy milk, tofu and tempeh, and theonsumption and use of soybeans (texturized soy protein, concen-rated soy protein and soy protein isolate) as additives by the foodndustry is increasing every year [1–4].

The potential of soybeans as a functional food is being currentlyxplored by the food industry. Indeed, soybeans and soy foods, likeoymilk, tofu, miso and tofu, are widely promoted and eaten basedn assumed relationships between its consumption and benefi-ial health effects in humans including chemoprevention of breastnd prostate cancer, osteoporosis, cardiovascular disease as wells relieving menopausal symptoms. Evidence provided not only bypidemiological studies showing a lower incidence of these healthonditions in Asian countries like Japan and China, which have highoy consumption, but also from intervention studies, is the basis ofhis relationship [5–12].

During the last decades our knowledge about the dietary impactn health and well-being has been highly increased and oftenelated to specific food components. Several classes of phytochemi-als have been identified in soybeans, including protease inhibitors,hytosterols, saponins, phenolic acids, phytic acid and isoflavones13–16]. Of these, isoflavones are particularly noteworthy becauseoybeans are the only significant dietary source of these com-ounds. Isoflavone content in soybeans can range from 0.4 mg to.5 mg of total isoflavones per gram, which can be influenced byenetics, crop year and growth location [17–19]. More importantly,hese compounds have shown several in vitro and in vivo beneficialroperties consistent with the potential soybean effects on health.

There are several possible mechanisms of action by whichsoflavones may act on disease prevention, including estrogenic/nti-estrogenic activity, cell anti-proliferation, induction of cell-ycle arrest and apoptosis, prevention of oxidation, anti-nflammatory, regulation of the host immune system, and changesn cellular signaling [7,20–28]. The actual mechanisms in the humanrganism have not been fully established and metabolism may playn important role. Furthermore, besides of evidence of availablepidemiological or intervention studies and “in vitro” observations,here are several reports indicating that several of the specificotential soybean health benefits are linked to isoflavone intake8,29–32].

However, there is still controversy and an unanimous positionbout if isoflavones, other soy phytochemicals or components areesponsible for the health benefits of soy consumption is still farrom being reached. Because the data in humans are not conclu-ive for any of these possible benefits, it is important to conductore studies investigating isoflavones and soy foods in the diet to

ealth outcomes. An accurate food composition database is cru-ial for such studies. That is the reason why there is an increasingnterest of scientists focused in developing newer extraction andnalysis methods for the characterization of soybean functional

omponents, especially isoflavones, and about the relationshipsetween their consumption and beneficial health effects inumans.

Isoflavones are a subclass of flavonoids and are also describeds phytoestrogen compounds, since they exhibit estrogenic activ-

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Fig. 1. Chemical structures of soybean isoflavones and abbreviations.

ty (similar effects to estradiol hormones). The basic characteristicsoflavone structure is a flavone nucleus, composed by two ben-ene rings (A and B) linked to a heterocyclic ring C (Fig. 1). Theenzene ring B position is the basis for the categorization ofhe flavanoid class (position 2) and the isoflavonoid class (posi-ion 3). The main isoflavones found in soybeans are genistein4′,5,7-trihidroxyisoflavone), daidzein (4′,7-dihidroxyisoflavone),lycitein (4′,7-dihidroxy-6-metoxi-isoflavone) and their respectivecetyl, malonyl and aglycone forms (Fig. 1) [33–39]. Biochanin A andormononetin (which are derivatives of genistein and daidzein) areenerally less abundant in soy than the 12 main forms and whichre found mostly in clover and alfalfa sprouts [40].

Isoflavone content of available soy foods in several countriess been intensively investigated. Quantification of isoflavones inhe soybeans and soy foods consumed in the USA [40–44], Japan45,46], Italy [47], UK [48,49], Singapore [43,50], Australia [51],ndonesia [50,51], Brazil [52], and Canada [53] have been publishedn the last decade.

Besides of individual reports, there are food compositionatabases and compilations from these values specifically focus-

ng on isoflavone distribution [54–62]. These reports supply usefulnformation to investigators determining the intake of phytoestro-ens in order to relate intakes to potential biological activities. Also,

hey can be used by health professionals and consumers to estimatendividual phytoestrogens intake and design personalized diets inrder to achieve biologically active concentrations of these func-ional compounds.

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When the intake of isoflavones is estimated, the quality of theood composition database is important. This is critical in the casef foods consumed regularly, in large quantities, or containingngredients with concentrated amounts of phytoestrogens. Futurenalyses of the isoflavone content of basic ingredient foods andommercial items commonly consumed in the diet will enableore accurate estimates of phytoestrogen intake and obtain reli-

ble conclusions about their role on health [56,58,63].Due to the enormous efforts done in the last few years to evalu-

te isoflavone composition in foods and its relation with nutritionalssues and health effects it is of ultimate importance to develop reli-ble and precise methods for the quantification of these compoundsn foods. Because of the increasing complexity of the food supply,here are major challenges in collecting reliable food consump-ion data for phytoestrogen intake estimates. Several extraction

ethods have been used for quantification purposes without ade-uate validation of the extraction procedure and far from optimizedxtraction conditions, which can lead to erroneous measurementsnd calculations. Besides, optimal extraction conditions can be usedo save time, resources and provide reliable information. More-ver, only scattered data are available in the scientific literaturend a review of the subject is needed to provide essential infor-ation on the topic and to identify future research fields of action.

herefore, the aim of the present manuscript is to provide a crit-cal review of the actual state, the most recent advances as wells current trends and future prospects in sample preparation andnalysis for the quantification of isoflavones from soybeans andelated foods.

. General aspects of soy isoflavones determination

The four common steps for any analytical method are sam-ling, sample preservation, sample preparation and analysis. Fig. 2resents a general overview of the most common steps for samplereparation for the determination of soy isoflavones.

The initial step in any analysis is sampling, where a representa-ive sample is collected from the entire sample matrix that needso be analyzed. The entire food-stuff should be represented in theample that will be used for the analysis. Sample preservation isn important step as there is often some delay between sample

ig. 2. Most common steps for sample preparation for the determination of soysoflavones.

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ollection and/or preparation and analysis. Proper sample preser-ation ensures that the sample retains its physical and chemicalharacteristics from the time it is collected to the time it is ana-yzed.

Sample preparation may consist of multiple steps such asrying, homogenization, sieving, extraction of target compounds,re-concentration, hydrolysis and derivatization. Sample prepara-ion can seek several objectives: to increase the efficiency of anssay procedure, to eliminate or reduce potential interferences, tonhance the sensitivity of the analytical procedure by increasinghe concentration of the analyte in the assay mixture, and some-imes to transform the analyte of interest to a more suitable formhat can be easily separated, detected, and/or quantified. Isoflavoneetermination is complex since its concentration in the sampleepends of several variables which may difficult the determination.verall, the ultimate goal is to obtain a concentrated extract withll isoflavones and free of interfering compounds from the matrix64–66].

The quantification of isoflavones in solid samples is usually per-ormed by extracting isoflavones from the food matrix using aertain solvent and then analyzing the extract by one of the sev-ral analysis techniques available, including gas chromatography,igh performance liquid chromatography (HPLC) and immunoas-ay, among others. The most used analysis technique is, withoutoubt, reverse-phase HPLC using C18 based columns with waternd methanol or acetonitrile containing small amounts of acid ashe mobile phase.

The extraction phase is extremely important and the processill depend of analyte liberation from the matrix, which will allow

uantitative determinations of target compounds. Moreover, thextract should mimetic the original isoflavone composition androfile as much as possible. For the efficient extraction severalarameters should be defined like the solvent, temperature, samplemount and time.

Optimization of the extraction conditions is normally accom-lished using the classical one-variable-at-a-time method, in whichhe optimization is directly assessed by systematic alteration ofne variable, while the others are kept constant. Some authorsse experimental designs for the determination of interactionsetween parameters and selecting the most suitable extractiononditions while minimizing the number of experiments. In thexperimental design strategies the values of all the factors undertudy are varied in each assay in a programmed and rational way. Its thus possible to detect the influencing factors while the numberf trials can be kept to a minimum [67,68].

. Sample stability

In analytical practices, the importance of sample conservationust be emphasized. Indeed, if not carefully controlled can lead to

rrors that cannot be corrected afterwards since will consequentlyffect the outcome of the final analysis. Thus, the results obtained,nstead of being the source of information, can produce misinfor-

ation.Often too little attention is given to the handling of soybean, soy

oods or isoflavone extract samples after their collection and beforehe actual instrumental analysis. How and for how long differentamples can be stored to preserve their original isoflavone profiles particularly important since some isoflavones have a relativelynstable character. Chemical changes of isoflavone structures have

een reported to occur during the processing of soybeans andoy products. The most frequently observed chemical changes ofsoflavones during the processing are decarboxilation of malonyllucosides to acetyl glucosides and ester hydrolysis of malonylnd acetyl glucosides to underivatized glucoside. It is also possible

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 2–29 5

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or all different conjugated forms to generate the aglycone formsy cleavage of the glucosidic bond [69–73]. An overview of theost common possible degradation paths of soy isoflavones are

resented in Fig. 3. However, only there are only a few studiesbout isoflavone stability during storage of soybeans, soy foods andxtracts. In fact, only recently the stability of isoflavones in soybeanstored under different conditions was investigated [74–76].

Information from the few reports available indicates that storagef soybeans and soy foods for prolonged times at room temperaturean affect isoflavone distribution and content. Generally, the con-entrations of individual isoflavones either significantly decreaser increase during storage for long periods. With storage, malonyllucosides concentration tends to decrease while concentrationf glucosides and aglycones tend to increase. Concentration ofalonyl glucosides can decrease by about 2 times, whereas glu-

osides and aglycone concentration can increase up to 3–4 timesuring storage for 2 years [74]. However, storage at room temper-ture may, in some cases, decrease glucoside and aglycone content75].

Moreover, not only the isoflavone profile may be affected by theourse of time, but also total isoflavone concentration, especiallyn the first year of storage. Afterwards, storage (up to 2 years) onlylightly changes total isoflavone content but still affects isoflavonerofile of the samples [74]. Storage for prolonged periods reducesotal isoflavone concentration and the reduction level dependsf the soybean cultivar. While some cultivars show only a slightecrease on total isoflavone concentration, others present a severeecrease on concentration of these compounds [75]. Furthermore,he level and type of the modification on isoflavone profile andosses caused by storage may be dependent of temperature, relativeumidity and the soybean cultivar among other factors.

The variation on isoflavone concentrations were positively cor-

elated with storage temperature and total isoflavones were relatedith the amount of malonyl glucoside and glucoside groups. Stor-

ge at low temperature can result in changes in isoflavone levelsimilar to those observed during processing [75] or may not affectsoflavone distribution [76]. Endogenous glucosidases, humidity

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ion paths of soybean isoflavones.

nd influence of soybean variety as observed by Kim et al. [75] may,artially, explain differences observed in these studies.

Relative humidity as well as temperature can influence thehanges on isoflavone profile during storage. Storage of soy-eans under high relative humidity (84%) and temperature (30 ◦C)onditions for extended periods of time (9 months) causes the inter-onversion between aglycones and �-glucosides. Storage underhese conditions can affect isoflavone profile to a point thathe major constituents can become the minor constituents, andice-versa. Storage under milder storage conditions (57% relativeumidity and 20 ◦C) causes only the interconversion between �-lucosides and malonyl glucosides [76].

It has also been demonstrated that some isoflavones in soymilkre subjected to degradation [77] during storage. For example, Gi isabile to degradation during storage at room temperature, althought a low rate. Losses of Gi with time showed typical first-order kinet-cs and increased with storage temperature. The Di concentrationas not influenced by storage between 15 ◦C and 37 ◦C. However,egradation of Di was not discarded, since it was possible thatcombination of deacetylation of ADi to Di and Di degradationas taking place simultaneously. At early stages of soymilk stor-

ge at 80–90 ◦C, ADi concentration increased, followed by a slowecrease. However, malonyl isoflavones, which are more sensitiveo degradation, were not studied.

Therefore, more research it is still needed on the effects oftorage environments, such as humidity and temperature, on theransformation and losses of isoflavone groups. The characteriza-ion of the differences between soybeans cultivars related withhe change of isoflavones, with special emphasis on endogenouslucosidases and to identify suitable conservation methods aremportant pending tasks. Also, more research aimed at differentoy products is required in the same direction.

Finally, it is imperative that authors conducting quantificationtudies be specific about sample conservation aspects. It must belear for how long the soybean or soy food sample have been storedefore actual analysis and the conditions such as temperature,umidity, etc.

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Also, while studying isoflavone profiles and distribution inoods and over different cropping years it is highly recommend-ble to perform the determinations after harvest, and not analyzell the samples at the same time (after all samples were har-ested), with the inherent differences and errors caused by storagefter 1 year or more, even if samples are stored at low tempera-ure. It is also recommendable to refer isoflavone content to dryeight since variation of sample humidity may influence concen-

ration.On the other hand, storage of samples after extraction and before

nalysis can also affect isoflavone profiles and result in avoidablenalytical errors. Due to the relatively unstable character somesoflavones as well as by the action of native �-glucosidases, result-ng in a rapid degradation or interconversion between chemicalorms, quantification of isoflavones is a complicated procedure. The

ost susceptible to degradation isoflavones seems to be the mal-nyl forms. Barnes et al. [78] noted that isoflavones in 80% MeOHxtracts of soy samples kept at room temperature were convertedradually from malonyl glucosides to �-glucosides. Coward et al.71] reported a slight conversion of the malonyl glucosides to the-glucosides conjugates at room temperature and that malonyl glu-oside conjugates are stable at 4 ◦C for 24 h, but prolonged storagelso causes conversion to the �-glucosides conjugates.

Later, Murphy et al. [42] reported a conversion rate of.2–0.3 mol% per hour of malonyl forms to glucosides in soy

soflavone extracts at room temperature. Evidences show thatrompt analysis of the extracts after extraction or other strategies,uch as maintenance of auto sampler at low temperatures (4–5 ◦C)re necessary to minimize degradation of malonyl isoflavones.

Although these procedures can elude potential analysis errors its essential to consider the stability of soy isoflavones extracts undertorage conditions to allow better planning of routine analysis ofarge number of samples and avoid analytical errors due to degra-ation and conversion between forms (i.e. malonyl to glucosides,alonyl to acetyl glucosides, etc.) [79].In one of the few published reports dealing specifically with the

torage of soy isoflavone extracts, Rostagno et al. [79] evaluated thenfluence of several factors (temperature, storage time, head spacend UV light) on short-term stability of samples kept on HPLC vials.he conclusion was that samples can be stored up to 1 week witho significant degradation if kept at temperatures lower than 10 ◦Cnd protected from light.

On the other hand, Rijke et al. [80] evaluated the stability ofsoflavone extracts obtained from red clover and observed that sam-les can be stored up to 2 weeks at −20 ◦C and if samples are keptt room temperature or if are stored dry at −20 ◦C, degradationtarts almost immediately. Curiously, they also observed that in LCeparated fractions, red clover malonyl isoflavones are more stablehen stored at low temperature after evaporation to dryness.

Aside the fact that the report did not include most commonsoflavones present in soybeans it indicates that more research iseeded to find more suitable sample conservation methods and tovaluate longer storage of soy isoflavone extracts under differentonditions before analysis.

. Hydrolysis

As previously discussed, there are different isoflavone chemicaltructures, and interconversion can occur between forms depend-ng of storage, processing and extraction conditions. Not only

ample preparation is complicated, but also the analysis step maye critical. The accurate quantification of the total content of

soflavones is hampered by the feasibility of chromatographicallyeparating all the possible forms of these compounds and to findhe corresponding reference standards. Some isoflavones are par-

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togr. A 1216 (2009) 2–29

icularly difficult to separate from each other (i.e. MGi, AGly ande) [81], while others (i.e. malonyl and acetyl isoflavones), due

heir relative unstable character, are not widely commercially avail-ble. Coelution of other substances present in the extracts may alsodd difficulty to the troublesome determination of soy isoflavones.urthermore, some isoflavones might occur in as yet unidentifiedorms.

A possible solution to these analytical problems is to performdequate sample treatment involving hydrolysis in order to reducehe number of isoflavone chemical forms occurring in the sample.he hydrolysis procedure itself can be carried out before, duringr after the extraction using different conditions and agents. Therere two main procedures to perform the hydrolysis of isoflavoneseported in the literature, basic or acidic hydrolysis. Basic hydrolysisct on ester bonds, removing acid groups that are linked to the sugaroiety of the isoflavone glucosides. As a result, the malonyl and

cetyl glucoside isoflavone forms are converted to their respectivelucosides. Acid hydrolysis breaks the bond between the isoflavonend the glucoside moieties, transforming all the isoflavone deriva-ives, into their aglycone forms [82].

Although reaction times and temperatures for the acidic hydrol-sis conditions vary a great deal, these procedures usually involvereating the extract or food sample itself with inorganic acid (HCl)t high temperatures in aqueous or alcoholic solvents with reactionimes ranging from a few minutes to several hours. Basic hydrolysisntails treating the sample with a solution of NaOH and allowingtanding at room temperature from a few minutes to overnight82–87].

Hydrolysis through the use of enzymes or a combination ofnzyme and acid [88,89] has also been used, although theseethods are less frequently used than acid or basic hydroly-

is. The enzymatic hydrolysis consists of incubating the sampleith enzymes for long periods of time, ranging from a few hours

o overnight. Different enzymes have been used for the hydrol-sis of isoflavones, including endougenous soy �-glucosidases,-glucuronidases, sulfatases and cellulases. Similarly to basic andcid hydrolysis, conditions vary a great deal and several differentethods have been reported [88–93].There are advantages and disadvantages with the use of

ydrolytic methods. The most obvious disadvantage is the inclusionf an additional step, with the inherent complication of the samplereparation procedure and the possible added analytical variabil-

ty. Also, there is indication that Ge is not entirely stable under acidydrolysis conditions [93]. The limited information obtained usingydrolytic methods can also be decisive, since only a few chemi-al forms are quantified, while using non-hydrolytic methods fullnformation can be accessed.

Although the hydrolysis step creates new questions with respectith sample preparation, analyte stability and recoverability, it

reatly simplifies the analysis by reducing the number of deriva-ives. The chromatographic analysis time is considerably shorternd separation of target compounds is easier since there are fewerompounds occurring in the sample. Acid hydrolysis results in thenclusion in the quantification of isoflavones that are linked to sug-rs other than glucose, and of glucosides of isoflavones that areot commercially available or difficult to acquire. Acid hydrolysis

s useful for the analysis of complex samples, and may be usedo identify sugar-isoflavones by comparison of these results withhose from basic hydrolysis. The analysis of acid hydrolyzed extractss preferred when analyzing samples of unknown origin, because

t includes in the quantification the glucoside derivatives of allsoflavones available only as aglycones [82].

Moreover, the use of hydrolytic methods may reduce the analyt-cal variability caused by stability issues during extraction since the

ost unstable isoflavones (malonyl glucosides) are not quantified

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s such. However, it is of crucial importance when using hydrolyticethods that authors make the necessary corrections and normal-

ze the results by molecular weight to the aglycone forms, since theolecular weight of the glucosides is greater than aglycones, and

herefore reported total isoflavone amount can be significantly lesshan the value of non-normalized data.

Although the available evidence in the literature suggests thathe biological effects of soy isoflavones depend upon aglycone form,or analysis of soy foods for isoflavonoids, the recent trend haseen to avoid hydrolysis. Using non-hydrolytic methods providealuable information about the exact distribution of all chemicalorms present in soybeans and soy foods. The different isoflavones

ay have differing pharmacokinetics and bioactivity [94–96] andhis may be a key factor in understanding their biological effects.nother logical reason to avoid hydrolysis is to minimize sampleandling, simplifying the extraction and overall analytical proce-ure and to shorten, as much as possible, the time required fromampling to actual analysis. However, it is important to highlighthat the valuable information about the total isoflavone concen-ration provided by hydrolytic methods is an essential screening

easurement and that isoflavone profiles are very important in andvanced metrological step.

. Extraction techniques and methods

.1. Solid and semi-solid samples

Optimal solid–liquid extraction involves the intimate contactetween a solid material, usually finely grinded, and a solvent thatas a maximal solubility for the analyte of interest and a minimalolubility for the matrix, using additional external forces and heat-ng to speed up the extraction process. Solid soy samples, such asoybeans and soy protein, require only grinding before extraction,ut sometimes are freeze-dried to provide a homogenous powder.iquid samples are most often freeze-dried and also treated as solidamples. Common methods for the extraction of the isoflavonesrom solid samples include organic solvent extraction with pure orqueous methanol (MeOH), ethanol (EtOH), acetonitrile (MeCN) orcetone (ACE) with and without the addition of small amounts ofcids using simple soaking, mixing, shaking or soxhlet extraction.he extraction time may range from 2 h to 24 h and the extractionemperature from 4 ◦C to 80 ◦C.

More recently, “modern” extraction methods, such as ultrasoni-ally assisted extraction (UAE), pressurized liquid extraction (PLE),upercritical fluid extraction (SFE) and microwave-assisted extrac-ion (MAE) have been used for the extraction of soy isoflavonessing similar solvents. In many cases, besides of filtration and cen-rifugation, further purification and/or pre-concentration of thearget compound fraction is applied. In these cases, evaporationo dryness and re-dissolution on another solvent or solid phasextraction (SPE) are the most commonly used methods. Anotherommon procedure during sample preparation is the hydrolysisfter the extraction (see Section 4).

.1.1. Conventional extraction methodsAmong the conventional extraction techniques soxhlet, shak-

ng, and stirring are the most commonly used for the extractionf isoflavones from soybeans and soy foods. There are numerousvailable extraction methods using these techniques with differ-nt conditions, and most of them without an appropriate method

ptimization.

Several parameters can influence the extraction of organic com-ounds such as polarity and amount of the solvent, temperature,ass and kind of sample and extraction duration. In the spe-

ific case of isoflavones, optimum solubility of the analyte in the

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togr. A 1216 (2009) 2–29 7

xtraction solvent, one of the key parameters of any extractionethod, is very difficult to achieve since there are several chem-

cal forms with different solubility coefficients in a given solvent.ost methods so far developed evaluate different solvents trying

o reach an optimal condition where extraction of all isoflavones isaximized. Although abundant research on soy isoflavones quan-

ification is available only a few reports deals with the developmentnd optimization of extraction methods for quantification studies.n overview of developed methods and evaluated parameters usingonventional techniques for the extraction of isoflavones from soy-eans and soy foods is presented in Table 1.

One of the first studies about the extraction of soy isoflavone wasublished by Eldridge [97], where pure MeOH and EtOH and withifferent water proportions, Ethyl acetate and MeCN were evalu-ted for refluxing extraction of isoflavones from defatted soybeans.rom the evaluated solvent systems, 80% MeOH gave the high-st isoflavone extraction yields and the most reproducible results.sing this solvent, 4 h seemed to be sufficient for extracting the

soflavones from soybean meal and no significant differences inhe extraction efficiency was reported when using different sol-ent:sample ratios (14:1 and 45:1). Once extraction conditionsere established, the method was used for the determination of

soflavones from soybean flours, protein concentrates and isolates.he same method was also used for the study of the effect of envi-onment and variety on the composition of soybean isoflavones98].

Another pioneer study about the extraction of isoflavones wasarried out by Murphy [99], who compared several solvents sys-ems (MeOH, ACE, MeCN, and chloroform-MeOH) for the extractionf isoflavones from toasted defatted soy flakes using wrist-actionhaker. The results indicated that extraction with pure solvents gaveow yields and that the addition of water or acid greatly improve thextraction efficiency of all isoflavones examined (Gi, Ge, Di and De).n terms of total isoflavones and coextractives, MeCN with water orcid was more efficient for the extraction of isoflavones all other sol-ents systems examined and no marked difference between thesewo solvents was observed in terms of total isoflavones.

As a result of these two pioneer studies, 80% methanol and acid-fied 83% acetonitrile became the most commonly used extractionolvents in isoflavone analysis. The method developed by Murphy99] has been extensively used with slight modifications in sam-le amount, solvent volume, addition of water to the extractingolvent and shaking technique [17–19,41,42,72,74,100–103]. How-ver, these slight modifications of the method have an importantmpact on extraction efficiency and should not be used lightly, sincextraction conditions require optimization for each different sam-le.

As an example, Song et al. [101] reevaluated the method by Mur-hy [99] and reported that using water in addition to HCl and MeCN

ncreased recovery. For different soy samples different amountsf water may be necessary maximize isoflavone extraction. Forost soy foods, 7 mL of water was sufficient to maximize extrac-

ion using a solvent volume:sample ratio higher than 6 mL g−1.t was also recommended that the solvent volume:sample ratiohould be adjusted for soy products with high concentration ofsoflavones, particularly for isoflavone supplements, which have

ore than 10 mg isoflavones/g. These investigators gave the exam-le of soy germ, which have high isoflavone content (>10 mg g−1),nd reported that the normal extraction procedure would underes-imate the isoflavone content by 10–20%. They found that adjusting

he ratio of solvent to sample weight to 95 mL g−1 resulted in morefficient extraction of isoflavones from the soy germ sample.

Following the evidence of the effect of the amount of water ofhe extraction solvent on isoflavone extractability, Murphy et al.42], reevaluated the same method and confirmed that adding a

8M

.A.Rostagno

etal./J.Chrom

atogr.A1216

(2009)2–29

Table 1Developed methods and evaluated parameters using conventional techniques for the extraction of isoflavones from soybeans and soy foods.

Sample used for evaluation of the method Isoflavones Fixed extraction conditions Evaluated parameters Selected conditions Reference

Defatted soybeans Di, Gi, Gly, De, Ge andGle

Solvent:

80% MeOH, 4 h [78]

Technique: refluxing EtOH, 50% EtOH, 80% EtOHSample: 1 g MeOH, 50% MeOH, 80% MeOHSolvent: 25 mL CH3CNTemperature: boiling point ofsolvent

Ethyl acetate

Extraction time: 1–5 h

Technique: Wrist-action shaker Solvent:Sample: 5 g MeOH, 80% MeOH, 80% MeOH

(HCl)Toasted defatted soy flakes Gi, Ge, Di and De Solvent: 25 mL of pure solvent

or: 5 mL (H2O or HCl0.1N) + 20 mL (solvent)

Chroloform–MeOH (90:10),80% chroloform–MeOH (90:10),80% chroloform–MeOH (90:10)(HCl)

80% CH3CN and 80% CH3CN(HCl)

[99]

Temperature: RT CH3CN, 80% CH3CN, 80%CH3CN (HCl)

Extraction time: 2 h ACE, 80% ACE and 80% ACE(HCl)

Technique: StirringSample: 2 g, Solvent:

Soy isolate, tofu, soybeans and miso Ge, De, Gi, Di, Gly, MGi, MDi,MGly and AGi

Solvent: 12–22 mL (12 mLCH3CN + 2 mL HCl 0.1N + water)

Different amounts of water(0–10 mL) added to the solvent(CH3CN)

The amount of wateroptimized depending of thesample ranged from 5 mL to10 mL of water

[42]

Extraction time: 2 hTemperature: RT

Toasted soy flourDi, Gi, Gly, De, Ge, Gle,MDi, ADi, MGi, AGi andMGly

Technique: tumbling mixer Solvent:1 h,RT

[73]Sample: 0.5 g 80% MeOH and 80% CH3CN

(0.1% HCl)Solvent: 4 mL Extraction time: 1, 2 and 24 h

Temperature: RT, 60 ◦C and80 ◦C

Technique: rotary mixer Solvent:Sample: 1 g or amountcontaining 10 mg totalisoflavones (always less than1 g)

10 mL CH3CN + 6 mLH2O + 0.5 mL DMSO (IS)

Soy protein Di, Gi, Gly, De, Ge, Gle, MDi,MGi, MGly, ADi, AGi and AGly

Solvent: ∼17 mL 10 mL CH3CN + 2 mL HCl0.1 M + 5 mL H2O

10 mL CH3CN + 6 mLH2O + 0.5 mL DMSO (IS)

[104]

Extraction time: 2 h 80% MeOHTemperature: RT Water % (10–100% CH3CN)

Technique: Stirring Solvent:Sample: 2 g 53% CH3CN, 53% ACE, 53%

EtOH, 53% MeOHSoy flour, tempeh, TVP and soy germ Di, Gi, Gly, De, Ge, Gle, MDi,

MGi, MGly, ADi, AGi and AGlySolvent: 19 mL (10 mLsolvent + 2 mL (HCl 0.1N orwater) + 7 mL water

With and without acid addition 53% CH3CN withoutacidification

[102]

Extraction time: 2 hTemperature: RT

Technique: Stirring Solvent:Sample: 2 g, 83% CH3CN, 83% CH3CN (+0.1N

HCl)

M.A

.Rostagnoet

al./J.Chromatogr.A

1216(2009)

2–299

Soybeans Di, Gi, Gly, De, Ge, Gle, MDi,MGi, MGly, ADi, AGi and AGly

Solvent: 12 mL 58% CH3CN, 58% CH3CN (+0.1NHCl)

58% CH3CN withoutacidification

[105]

Extraction time: 2 h 80% MeOH, 80% MeOH (+0.1NHCl)

Temperature: RT

Technique: Stirring Solvent:Sample: 0.5 g CH3CN (30–70%)

Freeze-dried soybeans Di, Gi, Gly and MGi Solvent: 25 mL EtOH (30–70%) 50% EtOH, 60 ◦C [106]Extraction time: 10 min MeOH (30–70%)

Temperature: 10 and 60 ◦C

Technique: Shaking Solvent:Sample: 2 g 80% CH3CN–HCl 0.1N

Defatted soybean meal, soy protein isolate Di, Gi, Gly, De, Ge, Gle, MDia,MGia and MGlya

Solvent: 10 mL 80% MeOH 80% CH3CN–HCl 0.1N, 5sequential extractions

[107]

Extraction time: 2 h 80% EtOHTemperature: RT Number of extractions: 1 and 5

Technique: homogenizationprobe and hand agitationSample: 0.1 g

Soybean flour Di, Gi, Gly, MDi, MGly, MGi, Deand Ge

Solvent: 4 mL (80% MeOH)(homogenization) + 1 mL(agitation)

Proposed method andreference method (modifiedMurphy method)

Proposed method [103]

Extraction time: 1 min(homogenization) + 30 min(agitation)Temperature: RT(homogenization) and 70 ◦C(agitation)

Soybean flour Ge and De

Technique: stirring Solvent: 40–99.99% EtOH99.99% EtOH,3:1 mL g−1, 80 ◦C and8 h

[20]Solvent: 4 mL (80% MeOH)(Homogenization) + 1 mL(agitation)

Volume:sample ratio: 1:1 to10:1 (mL g−1)

Extraction time: 1 min(homogenization) + 30 min(agitation)

Temperature: 40–90 ◦C

Temperature: RT(Homogenization) and 70 ◦C(agitation)

Extraction time: 2–24 h

De: daidzein, Ge: genistein, Gle: glycitein, Di: daidzin, Gi: genistin, Gly: glycitin, MDi: malonyl daidzin, MGi: malonyl genistin, MGly: malonyl glycitin, ADi: acetyl daidzin, AGi: acetyl genistin, AGly: acetyl glycitin, MeOH:methanol, EtOH: ethanol, CH3CN: acetonitrile, RT: room temperature.

a Tentatively identified by literature.

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ertain amount of water could optimize the total extraction. Extrac-ion conditions were optimized for each soy sample. The amount ofater had a significant effect of the amount of isoflavone extracted

nd varied with the food extracted. The amount of water optimized,epending of the food matrix, ranged from 5 mL to 10 mL (isolate,0 mL; tofu, 10 mL, soybeans, 7 mL, miso, 5 mL) using 2 g samples.

Also, the question of which extraction solvent is more efficients difficult to answer since it will depend of several factors suchs the technique, sample, amount of water, time, sample:solventatios, temperature, etc. For example, while Murphy [99] observedhat 80% MeCN (with and without acid) was more efficient than 80%

eOH (with and without acid), Barnes et al. [78] did not observeignificant differences between 80% MeOH and acidified 80% MeCNor the extraction of isoflavones from toasted soy flour toasted soyour using a different solvent to sample ratio.

Later, Griffith and Collison [104] proposed an improved proce-ure for the extraction of isoflavones from different soy samplessing 60% MeCN with 3% dimethylsulfoxide (DMSO) (v/v) andompared this solvent with 80% MeOH. This procedure was alsoompared with the modified Murphy [99] method used by Songt al. [101]. 80% MeOH was less efficient than MeCN (with andithout acidification + DMSO) in extracting most isoflavones andifferences between MeCN solvents (with and without acidifica-ion + DMSO) were smaller, with the primary difference in thextraction efficiency of more hydrophobic isoflavones (AGi, Ge, Dend Gle).

Afterwards, different water proportions of the extraction sol-ent were tested and 60% MeCN proved to be the most efficientolvent for two different soy protein samples (high and low in mal-nyl isoflavones). It was also observed an improvement (0.7–10.6%)n the extraction efficiency of different isoflavones from soy sam-les extracted with DMSO. The authors suggested that marginal

ncrease in isoflavone content might be attributed to the lack ofcid or to the presence of small quantity of DMSO. It is clear thatore research is still needed to evaluate the influence of DMSO

n extraction efficiency of isoflavones and examine the observa-ions reported in this study. Another interesting result was the smallffect of extraction time and the observation that the vast majorityf isoflavones were extracted in the first 5 min of extraction. Thiss strong evidence that the extraction time of similar methods cane drastically reduced from 2 h and this parameter can be furtherptimized.

Following the matter about the choice of the extraction solvent,urphy et al. [102], reviewed the extraction method and further

nvestigated MeCN, EtOH, ACE and MeOH in a 53% aqueous solu-ion with and without acid addition using the same method andoncluded that MeCN was more efficient than the other solventsnd that MeOH was the least efficient solvent in extracting the2 main isoflavone forms in raw soy flour, tofu, tempeh, textur-zed vegetable protein and soy germ. They also observed that theifferent solvents have different abilities to extract the different

soflavone forms and that the food matrix configuration may haven important impact on the extractability of the isoflavone forms.epending of the sample, some solvents may underestimate indi-idual isoflavone content up to 35% and total isoflavones up to 20%.nother important remark was that addition of acid reduced thextracted amount of some isoflavones and increased the extractionf others depending of the sample matrix. The authors suggestedhat in order to simplify the extraction protocol, it is probably betterot to use acid in the extraction medium for these food matrices.

n fact, the addition of small quantities of acid to the extractingolvent used by Murphy et al. [41,42,99,100] have been questionedince no clear differences or systematic pattern for all foods or forll isoflavone forms have been demonstrated and as evidenced byriffith and Collison [104].

tipov

togr. A 1216 (2009) 2–29

The initial purpose for the addition of small amount of acid waso increase the extraction efficiency and minimize coextractivesnd give clean HPLC chromatograms. However, in the initial report99], non-acidified MeCN extracted lower amounts of coextractivesith similar efficiency than acidified MeCN. Therefore, the use of

cidified MeCN seems not to make sense.Further evidence is provided by Lin and Giusti [105], who evalu-

ted the effects of solvent polarity and acidity on the extractionfficiency of isoflavones from soybeans. In this report, acidifiedolvents either extracted significantly (p < 0.05) lower amountsf isoflavones or did not significantly differ from solvents with-ut acid. Non-acidified solvents were more efficient in extractingalonyl isoflavones. For glucosides isoflavones, the acidification

howed a less significant effect on Gi and Gly and no relevant effectn Di. Also, no remarkable effect of acidification was found in thextraction of AGi and aglycones (Ge and De).

The differences in the total isoflavones obtained between acid-fied and non-acidified solvents mainly reflected the differences in

alonyl isoflavones. This may, in part, explain the results obtainedn the first report of Murphy [99] regarding the use of acidifiedolvents, since malonyl isoflavones were not measured in this study.

Moreover, a significant polarity–acidity interaction was foundor aglycone extraction, which suggests that the effect of the acidas not the same in the solvents with different polarities. Another

mportant observation was that acidification of the extraction sol-ent favored isoflavone transformations during the extraction andherefore should be avoided for quantification of intact isoflavones105].

Regarding the extraction efficiency of the solvents, results indi-ated that for all glucoside isoflavones the solvent with higherolarity (58% MeCN) either extracted significantly higher amountsr did not significantly differ from the assayed solvents with lowerolarity (80% MeOH and 83% MeCN). The differences between 58%eCN (most polar) and 83% MeCN (least polar) were important in

erms of extraction efficiency of individual and total isoflavones.owever, differences between 58% MeCN and 80% MeOH oretween 80% MeOH and 83% MeCN were not always relevant.

On average, 58% MeCN extracted significantly higher amountsf malonyl glucosides than 80% MeOH and 83% MeCN. Recover-es of aglycones, Ge and De with 80% MeOH resulted significantlyower than those obtained with the other evaluated solvents. Theifferences in measured isoflavones between solvents with variousolarities reflected the differences in malonyl glucosides, becausealonyl glucosides was the major form of isoflavones in the soy-

eans and it was most affected by solvent polarity.Therefore, solvents with relatively higher polarity and no acid

ere more efficient in general for extracting isoflavones. Amonghe six examined solvents, 58% MeCN without acidification washe best solvent for the extraction of isoflavones from soybeans,ince it yielded the highest total amounts and best maintained thentact structures. With regard to the two most widely used sol-ent systems, 80% MeOH had a higher extraction efficiency andetter protection against chemical transformation than acidified3% MeCN.

These results are in agreement with those reported by Rostagnot al. [106] who compared different solvents for the extractionf isoflavones glucosides and MGi from soybeans. These authorsbserved that when using pure solvents, low extraction effi-iency was obtained and that the maximum amount extracted wasbtained using solvents with 40–60% of water. They also observed

hat temperature has a great impact on the extraction efficiency ofsoflavones. Rostagno et al. [106] also reported that most isoflavonesresent in the sample (80–90%) were extracted in the first 10 minf extraction at 60 ◦C using 50% EtOH, corroborating similar obser-ations reported by Griffith and Collison [104].

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Another approach of solvent selection was given by Achouri etl. [107]. These authors compared three solvents (80% MeCN–HCl.1N, 80% MeOH and 80% EtOH) for the extraction of isoflavonesrom defatted soybean meal (DSM) and from soy protein isolateSPI). In the case of the DSM, the conclusion was that acidified 80%

eCN is more efficient for the extraction of malonyl isoflavonesnd aglycones, while 80% MeOH is more efficient for the extrac-ion of glucosides (using one extraction). In the case of the SPI, 80%tOH extracted the highest amount of aglycones, no significant dif-erence was observed between 80% EtOH and 80% MeOH for thextraction of glucosides and that acidified 80% MeCN extracted theighest amount of malonyl glucosides (using one extraction). How-ver, 80% MeOH extracted the highest amount of total isoflavones,ollowed by 80% EtOH and by acidified 80% MeCN in this particu-ar sample. These results indicate that the extraction efficiency ofhe solvent will depend of the sample from which the isoflavonesre extracted. One of the most interesting remarks in this reportas that individual amount of isoflavones extracted after the first

xtraction increased significantly after 5 consecutive extractions42–100% depending of the isoflavone) in soy meal, and in soyrotein isolate (89–153% depending of the isoflavone). For theifferent solvents used, the yield of total isoflavones after 5 extrac-ions (compared to only one extraction) increased between 65%nd 74% for the DSM sample, and increased by between 107% and47% for ISP sample, depending of the solvent. The most impor-ant observation in this report was that no significant differencen terms of total isoflavones was observed between the assayedolvent after 5 sequential extractions in the DSM sample indi-ating that it is possible to achieve quantitative extraction withny of the most commonly solvents used for isoflavones extrac-ion, given that conditions are optimized enough. Moreover, theseesults strongly suggest that one time extraction of isoflavone usingonventional methods markedly under-estimates the concentra-ion of isoflavones in these products.

However, sample characteristics are likely to play an importantole in the ability of a given solvent to extract isoflavone from soy-eans and soy foods. It is very interesting the observed variation

n the extraction yield of isoflavones between DSM and SPI. Forhe high protein sample (SPI), a unique extraction extracted only1% of total isoflavone compared to 58% of lower protein sampleDSM) using MeCN–HCl solvent. This difference was attributed totronger protein–polyphenol interaction in the SPI sample since aariety of interactions including hydrogen bonding, ionic and cova-ent binding, and mainly hydrophobic interactions are involved inhe formation of protein–polyphenol complex [108]. These inter-ctions are strongly influenced by factors such as temperature, pHnd salt, which occur during acidic precipitation of soy proteins.his outcome may also indicate that grinding and the resultingarticle size might, due to the effect in the matrix, can influencehe ability of the different solvents to extract isoflavones. The samerinciple can be extended to freeze-drying, which more severelyffect sample matrix structures.

Another extraction method using 80% MeOH for the analysis ofsoflavones from soybean flour was later proposed by Tsai et al.103]. The proposed method was compared with a modified Mur-hy method (using different sample to solvent ratio). They observedhat, except De and Ge, contents of detected isoflavones (Gi, Di, Gly,

Gi, MDi and MGly) extracted by the proposed method were higherhan those extracted by the modified Murphy method. These find-ngs imply that that several reports of isoflavone distribution in

oods using the method by Murphy are underestimating isoflavoneoncentration.

On the other hand, Zhang et al. [20] evaluated several extrac-ion conditions for the extraction and purification of isoflavonesrom soybeans. Extraction conditions included EtOH percentage

m

5Aa

togr. A 1216 (2009) 2–29 11

40–99.99%), solvent volume to sample ratio (1:1 to 10:1 mL g−1),emperature (40–90 ◦C) and extraction time (2–24 h). In this report,he influence of some extraction parameters was different thanhose obtained by other authors. Pure EtOH extracted the highestmount of isoflavones, while in most studies is clear that a certainmount of water (40–60%) in the solvent is necessary to improvextraction. Also, increasing solvent volume to sample ratio from 3:1o 8:1 (mL g−1) negatively affected yield. The objective of this studyowever, is the key to understand the differences on the effect of theample amount observed by other authors. In this case, the objec-ive was to extract the highest amount of aglycones and to obtainconcentrated extract, not to quantify all chemical forms. Usinghigher amount of sample with lower amounts of solvent vol-

me, it is logical that the concentration of isoflavone on the extractends to increase although lower relative extraction efficiency ischieved.

Summing up, the differences among the extraction methodseported in this review are most probably related with the amountf water used in the extraction solvent, the sample matrix, thextraction technique, sample to solvent ratio and more importantly,he isoflavone forms that were quantified. In some cases compari-on of extraction solvents were carried out for only a few isoflavonesresent. In this context, it is important to note that some chemi-al forms are responsible for the greater part of the total amountf isoflavones present in soybeans and soy foods, especially somealonyl and glucoside forms. Moreover, differences in analyticalethods and reporting of isomeric conversions can also contribute

ignificantly to variation on the results found in the literature. Inome studies, total isoflavone is expressed as the sum of all 12 iso-ers. In other studies, only aglycone and/or conjugated forms are

ested and expressed. Furthermore, in other studies isoflavones areydrolyzed to their aglycone forms or the amount is normalized byolecular weight to the aglycone forms. Also, some methods were

eveloped before the malonyl glucoside isoflavones were identi-ed [38,39] and therefore the results needed to be revised for thextraction of all 12 isoflavone forms. More importantly, the effectf extraction conditions on stability was not considered in manyases.

In general terms, the choice of the most appropriate solvent willepend of the isoflavone in highest amount present in the sam-le, since the most effective solvent for this particular isoflavoneill strongly influence the total amount extracted. For compari-

on purposes it is important to evaluate different solvents withoutchieving quantitative recoveries otherwise it will be impossibleo determine the magnitude of effect of the solvents. However, theecent trend is to avoid toxic and use environmental friendly sol-ents such as EtOH. EtOH can be highly effective for the extractionf isoflavone from soy samples with the advantage of lower cost,ower toxicity and environmental compatibility.

It also appears clear that to obtain quantitative extraction for thenalysis of the isoflavone content of foods is necessary to adjustxtraction conditions for each sample and some research is stilleeded to optimize other extraction variables, especially sample toolvent ratio and extraction time.

One of the important conclusions when reviewing informationvailable is that it is possible to achieve quantitative extrac-ion using most commonly used solvents and that it very likelyhat sequential extractions are required, as previously mentioned.inally, more research is needed to evaluate and explain the influ-nce of the sample, since it may be the answer to achieve standard

ethods for the extraction and analysis of isoflavones in foods.

.1.1.1. Stability during extraction using conventional techniques.part of optimizing extraction variables such as solvent, samplemount, temperature and duration, the assessment of stability

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2 M.A. Rostagno et al. / J. Ch

uring extraction is essential. Frequently, authors tend to overex-end extraction duration in order to achieve higher extractionields. This strategy, however, may cause not only degradation ofome chemical forms, but also the generation of other isoflavonesorms and isomers that can drastically modify isoflavone profile ofhe sample and influence the results obtained. Thus, although longxtraction times have been extensively used for the extraction ofsoflavone from soy and other matrixes, there are still several issueshat should be addressed such as the stability during extraction.

Extraction of isoflavones from foods or dietary supplementss a critical process since isoflavone profile can be altered dur-ng sample preparation since mild heat and acid are frequentlynvolved in the extraction, which could cause degradation of mal-nyl isoflavones and the hydrolysis of glucosides. Therefore, whenhoosing the extraction conditions it is important not only con-ider extraction efficiency, but also avoid, or at least minimize, thertificial transformations. Thus, temperature conditions during thextraction procedures as well as extraction duration have to bearefully adjusted because of possible degradation of the gluco-ide derivatives. Also, stability may be related to the solvent used,pecially acidified solvents.

One of the earlier observations of the influence of the extrac-ion temperature on the isoflavone profile was reported by Kudout al. [39]. They observed that malonyl isoflavone glucosides in0% alcohol extracts from both soybean hypocotyls and cotyledonsecreased significantly as their respective glucosides increasedhen the samples were extracted at 80 ◦C instead of room tem-erature.

The effects of extraction temperature on isoflavone profile wereater confirmed by Barnes et al. [78]. They observed that extractionserformed at 60 ◦C caused heat induced de-esterifying reaction ofalonyl and acetyl glucosides to their respective glucosides and

hat increasing temperature to 80 ◦C led to higher conversion rate.oreover, the changes on isoflavones profile were not only due

o temperature variations, but also time dependent. Even at roomemperature malonyl glucosides were gradually converted to theirespective glucosides. The conversion rate at room temperatureas later reported to be between 0.2 mol% and 0.3 mol% per hour

42]. Obviously, extraction methods using long extraction times canignificantly underestimate malonyl glucoside concentration andverestimate glucoside concentration.

Coward et al. [71] evaluated the effect of the temperature on thextraction of isoflavones from soy foods. Isoflavone �-glucosidesonjugates were extracted with 80% MeOH from soybeans at roomemperature, at 4 ◦C and at 80 ◦C, for 2–72 h by tumbling or shaking.uantitative and reproducible recovery of the isoflavone glucosidesas achieved after 2 h. Extraction at 4 ◦C gave the highest con-

entration of malonyl glucosides and the lowest concentration of-glucosides conjugates. Extraction at 80 ◦C caused extensive con-ersion of the malonyl glucosides conjugates to the �-glucosideonjugates but not to the acetyl conjugates or aglycones. Althoughhe composition of the individual �-glucosides was drasticallyltered by temperature, the total amount of isoflavones extractedas constant.

On another study, Franke et al. [93] evaluated the stabilityf De, Ge, coumestrol, formononetin, biochanin A and flavonender refluxing for 4 h using acidified 77% EtOH (2.0 M HCl) andbserved that only flavone was entirely stable. Therefore, it is clearhat refluxing is not recommendable for extraction of isoflavonesrom soybeans and soy foods, since it can cause losses, even if

ydrolytic methods are used. However, this may be related to these of acidified solvent as reported by Lin and Giusti [105], who

ater observed the transformation of �-glucosides to their cor-esponding aglycones and transformation of acetylglucosides toheir corresponding �-glucosides when subjected to extraction by

oamst

togr. A 1216 (2009) 2–29

tirring for 2 h at room temperature. Another important remarkas that acidification of the extraction solvent favored isoflavone

ransformations during the extraction and should be avoided foruantification of intact isoflavones.

Therefore, when evaluating an extraction method it is of cru-ial importance to know the stability of target compounds, in ordero maintain the isoflavone profile in the sample, unless hydrolytic

ethods are used (see Section 4). Submitting an extract obtainedith optimal conditions to the extraction protocol and compar-

ng concentrations is a simple way to perform such stability tests109,110]. It cannot guarantee that target compounds are entirelytable since other sample matrix components may influence sta-ility, but may give clues about the possible degradation underxtraction conditions. The use of extracts is preferable to the use oftandards since extracts contain other components and are morelose to real samples. Another method is to control concentrationf malonyl isoflavones trying to identify degradation patterns orse hydrolytic methods, quantifying aglycone equivalents. The usef an internal standard may also prove useful in this case.

The most recent trend regarding stability during extraction is tose of �-glucosidase inhibitors. Toebes et al. [111] identified Tris assuitable �-glucosidase inhibitor in red clover extracts, which wasptimized at 350 mM in 80% EtOH at pH 7.2. Extractions using Trisielded much higher amounts (13–24 times) of malonyl isoflavoness opposed to extractions without Tris. Although it was evaluatedor the extraction of isoflavones from red clover, the same prin-iple may be applicable for the extraction of soy isoflavones. Inhis case, however, concentration of Tris might need adjustmentnd further investigation, but unveils a strategy to avoid degrada-ion and, therefore, increase the reliability of results obtained in theuture. Other possible candidates for this role are HgCl2, AgNO3 and-glucono-ı-lactone, which have been reported to inhibit soybean-glucosidase, being the later the most potent inhibitor [112].

Although important advances have been made regarding thetability of isoflavones during extraction using conventional tech-iques, it is clear that more studies are necessary, especially withhe aim of avoiding degradation in order to provide reliable infor-

ation about the concentration of these compounds in foods. Also,ample and solvent characteristics have to be further examined inetail as well as other factors such as temperature and extractionechnique.

.1.2. Modern extraction techniques and methodsThe development and application of “modern” sample-

reparation techniques with significant advantages over conven-ional methods (e.g. reduction in extraction time, organic solventonsumption and in sample degradation, elimination of additionalample clean-up and concentration steps before chromatographicnalysis, improvement in extraction efficiency, selectivity, and/orinetics, ease of automation, etc.) for the extraction and determi-ation of isoflavones from soybeans and derived foods is playing an

mportant role in the overall effort of ensuring and providing highuality data for researches worldwide.

With this in mind, newer extraction methods have been devel-ped using modern extraction techniques, including supercriticaluid extraction, ultrasound-assisted extraction (UAE), pressurized

iquid extraction (PLE), microwave-assisted extraction and solidhase extraction.

When selecting the appropriate solvent for the extraction ofsoflavones using conventional extraction techniques, solubility is

ne of the most important factors. However, the selection of anppropriate solvent using “modern” extraction techniques is muchore complex, since it will depend of other factors besides of the

olubility of target compounds, such as the ability of the solvento absorb microwave energy (MAE), how it propagates ultrasonic

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aves (UAE) and the physical–chemical changes in the solvent thatake place under elevated temperature and pressure, which willlso affect solubility of target compounds (PLE/SFE).

.1.2.1. Ultrasound-assisted extraction. The enhancement of extrac-ion efficiency of organic compounds by ultrasound is attributed tohe phenomenon of cavitation produced in the solvent by the pas-age of an ultrasonic wave. Cavitation bubbles are produced andompressed during the application of ultrasounds. The increase inhe pressure and temperature caused by the compression leads tohe collapse of the bubble, resulting on a “shock wave” that passeshrough the solvent enhancing the mixing. Ultrasound also exertsmechanical effect. When a bubble collapses near a solid surface

t occurs asymmetrically and generates high-speed jets of solventowards the cell walls, therefore increasing the solvent penetrationnto the cell and increasing the contact surface area between solidnd liquid phase. This effect coupled with the enhanced mass trans-er and significant disruption of cells, via cavitation bubble collapse,ncreases the release of intracellular product into the bulk medium.he use of higher temperatures in UAE can increase the efficiencyf the extraction process due to the increase in the number of cav-tation bubbles formed. Several extraction parameters, similar toonventional extraction methods, can influence the extraction ofrganic compounds using ultrasounds, such as polarity and amountf the solvent, the mass and kind of sample and extraction timemong others. Also, parameters regarding the ultrasound sourceuch as frequency and intensity as well as the number of pulsespplied can have great impact on extraction dynamics [113–117].

Ultrasound-assisted extraction has been used in several occa-ions to extract isoflavones from soybeans, soy foods and fromifferent matrixes, such as Peanuts, Trifolium pretense, Puer-riae radix, Pueraria lobata, Radix astragali and Glycyrrhizae radix80,82,106,118–125]. However, optimization of UAE based meth-ds has not been conducted with a few exceptions. An overviewf the developed methods using ultrasounds for the extraction ofoy isoflavones and evaluated parameters is presented in Table 2.

One of the first methods where extraction conditions wereystematically assessed to achieve quantitative extractions of soysoflavones was published by Rostagno et al. [106]. For the methodevelopment, several extraction parameters were studied includ-

ng solvent, extraction temperature, sample amount and extractionime. The most important parameters affecting the extraction effi-iency were the extraction solvent (and the amount of water),xtraction temperature and extraction time. The extraction effi-iency was improved by using ultrasounds but was dependent ofhe solvent employed. 50% EtOH, 50% MeOH and 40% MeCN werehe solvent that extracted the highest amount of total isoflavonesith similar extraction efficiency. The best extracting solvent for

ach isoflavone form depended of the chemical form itself. Forll chemical forms best extraction efficiency was achieved usingolvents with 40–60% of water.

Extraction temperature had a great impact on the extraction effi-iency while using higher temperature significantly increased themount of all tested isoflavones. In general, the method was foundo be fast and reliable achieving quantitative extractions in 20 min.o be sure that quantitative recovery was achieved; results wereompared with 5 sequential extractions with no significant differ-nce. Most isoflavones (80–90%) occurring in the soy flour sampleere extracted in 10 min; corroborating the results obtained byriffith and Collison [104] (see Section 5.1.1). Extending the extrac-

ion length to 30 min decreased the yield of some isoflavones. Also,o significant difference was observed between ultrasonic probend ultrasonic bath and therefore, it can be used as an alternativeith the advantage of allowing the extraction of multiple samples.

he method developed by Rostagno et al. [106] has been used, with

aaimh

togr. A 1216 (2009) 2–29 13

nd without modifications, for routine analysis, to obtain isoflavonextracts for other studies and as reference method for comparisonf other extraction methods [80,82,109,110,119,126–138].

Regarding the extraction solvent, Achouri et al. [107] comparedhree solvents (80% MeCN + HCl 0.1N, 80% MeOH, 80% EtOH) forhe ultrasound-assisted extraction of isoflavones from different

atrixes (defatted soybean meal and from soy protein isolate)nd observed that 80% MeOH and 80% EtOH extracted the highestmount of isoflavones from both samples. They also observed thatonication for 15 min extracted as much as the total of 5 sequentialxtractions (with ordinary shaking for a total of 10 h), except forcidified MeCN. This is an important observation, since acidifiedeCN is one of the most used solvent with conventional extraction

echniques and points that it is not recommendable to use this sol-ent when using ultrasounds, since it can seriously underestimatesoflavone content of foods. It was also observed that extending theime of sonication from 15 min to 30 and 60 min, did not increasehe total amount of isoflavone extracted, and in some cases theotal amount decreased, corroborating the observations made byostagno et al. [106].

More recently, Bajer et al. [129] compared pure MeOH, MeCNnd ACE for the extraction of De and Ge from soy flour. MeCNave the highest yields and was further studied adding differentmounts of water (0–50%) and 60% MeCN gave the best results.emperature was also evaluated between 25 ◦C and 80 ◦C as wells extraction time between 10 min and 50 min. Highest amountsf isoflavones were obtained at 50 ◦C for 40 min using the ultra-onic bath. Also, using an ultrasonic homogenizer pulse generatoras evaluated in the range of 45–98 W (100%) the use of ultrasoniculses during extraction and the extraction time in the range of0–50 min. Best extraction yields were obtained using 60% of ultra-onic amplitude for 30 min. These results were obtained at roomemperature. In this report, unfortunately, information of the influ-nce of studied extraction conditions and their respective data wasot given, only a few isoflavones were studied and was limited inerms of the types of samples evaluated.

The influence of ultrasound on the solid–liquid extraction pro-ess as regards yields or selectivity is very difficult to predictecause of the interaction of many factors, either relative to thehase system (solid, liquid/solute) or to the ultrasonic reactor itself.he differences observed on the amount of water used in the sol-ent by the different reports may be related with the type of samplesed and its characteristics. It is very likely that the amount of watereeded to achieve maximum extraction efficiency might need somedjustment depending of the sample type, as reported by Murphyt al. [42] using conventional stirring.

Other factors may be influencing the extraction dynamics, sinceAE is affected by the ultrasonic wave distribution inside thextractor. Maximum ultrasound power is obtained at the vicin-ty of the radiating surface of the ultrasonic source and an abruptecrease of the ultrasonic intensity increases as the distance fromhe radiating surface increases. Furthermore, the presence of solidarticles can affect the ultrasonic intensity profile, which can bere attenuated depending of nature of the sample such as hardness,ompactness, particle size and solute distribution [130].

Also, the influence of other important extraction variables suchs frequency, intensity and the use of ultrasonic pulses have noteen extensively studied in detail and for all isoflavones and there-ore future investigations should focus on these issues as well as onhe influence of the sample on the extraction. In general, UAE seems

potent technique for the extraction of isoflavones from soybeansnd soy foods. This technique can achieve high extraction yieldsn less than 30 min from different sample types using the com-

only used solvent in conventional methods. It clear though, thatigh temperatures can be used since extractions are short and that

14 M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 2–29

Table 2Developed methods using ultrasounds for the extraction of soy isoflavones and evaluated parameters.

Sample used for evaluationof the method

Isoflavones Fixed extractionconditions

Evaluated parameters Selected conditions Reference

Freeze-dried soybeans Di, Gi, Gly and MGi

Solvent:

50% EtOH, 60 ◦C, 0.1 g, 20 min [106]

EtOH (30–70%)MeOH (30–70%)

Solvent: 25 mL CH3CN (30–70%)Vibrationamplitude: 100%

Temperature: 10 and 60 ◦C

Sample amount: 0.5–0.1 gExtraction time: 5–30 minUltrasound source: ultrasonic probe andultrasonic bath

Sample: 2 g Solvent:Solvent: 10 mL 80% EtOH

Defatted soybean meal andsoy protein

Di, Gi, Gly, De, Ge,Gle, MDia, MGia andMGlya

Temperature: 22 ◦C 80% MeOH 80% MeOH and 80% EtOH,15 min

[107]

Ultrasound source:ultrasonic bath

80% CH3CN (0.1N HCl)

Extraction time: 15–60 min

Solvent:EtOHMeOH

Sample: 1 g(ultrasonic bath)and 2 g (ultrasonichomogenizer)

CH3CN (50–100%) 60% CH3CN

Soy flour De and Ge Solvent: 25 mL(ultrasonic bath)and 45 mL(ultrasonichomogenizer)

Temperature: 25–80 ◦C (ultrasonic bath) Ultrasonic bath: 50 ◦C,40 min

[129]

Temperature: RT(ultrasonichomogenizer)

Extraction time: 10–50 min Ultrasonic homogenizer:30 min, and 60% vibrationamplitude, pulse generator(not specified)

Ultrasound source: ultrasonic bath andultrasonic homogenizerPulse generator: 45–98 WVibra

D Di: mA aceto

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e: daidzein, Ge: genistein, Gle: glycitein, Di: daidzin, Gi: genistin, Gly: glycitin, MGi: acetyl genistin, AGly: acetyl glycitin, MeOH: methanol, EtOH: ethanol, CH3CN:a Tentatively identified by literature.

ntermediate to high amounts of water in the extraction solvent40–80%) are needed to efficiently extract isoflavones.

The influence of ultrasounds on isoflavone distribution dur-ng extraction should not be neglected. The same principles ofsoflavone stability during extraction using conventional tech-iques apply when using ultrasounds. However, there are other

actors which can affect stability of these compounds such as theroduction of radicals from the ultrasound dissociation of water.

n the presence of these high energy species, oxidative reactionsan take place simultaneously with the extraction reactions whenater is higher than 50% [106]. This is particularly important, since

s previously mentioned intermediate to high amounts of watern the extraction solvent (40–80%) are needed to efficiently extractsoflavones.

Rostagno et al. [106], for example, observed a reduction of thextraction efficiency common to solvents with high amounts ofater (>60%) which was attributed to an increased production of

adicals from the ultrasound dissociation of water. In such reportlightly lower yields of total isoflavones were obtained using extrac-ions of 30 min with 50% EtOH at 60 ◦C than those obtained with

xtractions of 20 min. Similar results were obtained by Achouri etl. [107], who observed that, in some cases, the total amount ofsoflavones decreased if extraction were extended from 15 min to 30nd 60 min. In view of this evidence it seems advisable to use shortxtractions rather than long extractions when using ultrasounds.

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tion amplitude: range not specified

alonyl daidzin, MGi: malonyl genistin, MGly: malonyl glycitin, ADi: acetyl daidzin,nitrile, RT: room temperature.

Stability of isoflavones during UAE has not been apparently stud-ed to the moment. With the available evidence that relatively shortxtraction times can affect isoflavone profile and content, assess-ent of the influence of ultrasounds on isoflavone degradation is

f one of the most urging needs in future research in this field. Theffect of extraction solvent, temperature, ultrasound intensity andrequency on stability of soy isoflavones during UAE and the searchf effective ways to avoid degradation need to be further examinedn detail in future investigations.

.1.2.2. Pressurized liquid extraction. Pressurized liquid extractions a procedure that combines elevated temperature (50–200 ◦C)nd pressure (100–140 atm) with liquid solvents (without theirritical point being reached) to achieve fast and efficient extrac-ion of the analytes from the solid and semi-solid samples matrix.his technique has received different names, such as acceleratedolvent extraction (ASE), pressurized liquid extraction (PLE) andressurized solvent extraction (PSE). When water is employed ashe extraction solvent, the authors tend to use a different name,uch as superheated water extraction (SWE), to highlight the use

f this environmental-friendly solvent.

For rapid and efficient extraction of analytes from solid matri-es, extraction temperature is an important experimental factor.levated temperatures can lead to significant improvements inxtraction efficiency, since it may increase solubility of target

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M.A. Rostagno et al. / J. Ch

ompounds, diffusion rates and mass transfer of analytes to the sol-ent. Moreover, temperature can dramatically modify the relativeermittivity of the extracting solvent, increasing selectivity. Highressure allows maintaining the solvent in a liquid state at highemperature and may increase the penetration of the solvent in theample matrix. Extractions performed under elevated temperaturend pressure results in adequate kinetics of dissolution processesnd favors desorption of analytes from the surface and active sitesf solid sample matrices [67,113,114,131,132].

PLE has been used in several occasions to extract isoflavonesrom soybeans, soy foods and other different matrixes such as Radixuerariae, Matricaria recutita, Rosmarinus officinalis, Foeniculum vul-are and Agrimonia eupatoria L. [109,121,129,133–140]. An overviewf the developed methods using pressurized liquids for the extrac-ion of soy isoflavones and evaluated parameters is presented inable 3.

The same principles of isoflavone stability during extractionsing conventional techniques also apply when using PLE. The usef high temperatures can strongly affect isoflavone content androfile as previously discussed in Section 3. In the case of PLEowever, temperatures used are much higher than those used inonventional methods and thus it can expected that the extend ofegradation and transformations taking place during extraction areuch more important.Rostagno et al. [109] evaluated the influence of several extrac-

ion parameters, such as solvent, temperature, pressure, sampleize, static extraction cycle length and number of static extrac-ion cycles in order to optimize extraction conditions to achieveuantitative recoveries of isoflavone from freeze-dried soybeans.hey observed that using EtOH/water mixtures, extraction effi-iency increased when increasing the water percentage in thextraction solvent from 0% to 30%, and that higher amount ofater in the extraction solvent resulted in a lower extraction

fficiency. Similar results were obtained for MeOH/water mix-ures, and the highest extraction efficiency was achieved using 60%

eOH. Water extracted the lowest amount of isoflavones betweenssayed solvents. They also reported that increasing the extrac-ion temperature from 60 ◦C to 150 ◦C increased the yield of mostsoflavones (except the malonyl forms) and identified a degrada-ion pattern. The increase in the extraction temperature from 60 ◦Co 100 ◦C increased the total amount of isoflavones extracted inpproximately 20%. The increase of the yield of isoflavone glu-osides with the increase of temperature between 100 ◦C and50 ◦C was very pronounced (approximately 30%) while the yieldf malonyl isoflavones decreased (approximately 50%), when itas expected to follow the same trend as the glucoside forms and

ncrease.Searching for answers, a stability evaluation of extraction con-

itions was performed which confirmed that degradation startsbove 100 ◦C for the malonyl forms and above 150 ◦C for thesoflavone glucosides. Above 100 ◦C, with the decrease of MGi, aorrespondent increase in Gi concentration was observed. Con-entration of other glucosides also increased at this temperature.glycone levels remained constant below 150 ◦C indicating thategradation of glucosides was not taking place below this tem-erature. Above 150 ◦C, aglycone levels showed a small increaseith the decrease in their respective glucoside levels, indicating the

onversion between these chemical forms. The stability study con-rmed the observations made during the extraction temperatureptimization, indicating that 100 ◦C is the maximum temperature

or PLE of isoflavones. It was also reported that the increase of pres-ure from 100 atm to 200 atm did not have a significant impact onhe extraction of isoflavones from freeze-dried soybeans, and thateducing sample size (from 0.5 g to 0.05 g) increased the yield ofsoflavones in approximately 13%. However, relative standard devi-

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togr. A 1216 (2009) 2–29 15

tion increased proportionally. The extension of the three staticxtraction cycle used from 5 min to 7 min increased the extractionield in approximately 10% and no significant effect was observedetween 7 min and 10 min. To ensure that quantitative extractionas obtained, the authors performed four re-extractions of the

ample achieving similar recoveries.With the evidence provided by this report, it is clear that caution

hould be used when increasing the extraction temperature andhat more research is needed to evaluate the stability of isoflavonesuring PLE. Among the main factors that should be studied in detail

n future researches are the influence of the sample, solvent and theuration of the procedure.

An interesting method for the extraction of isoflavones from soy-ean foods was developed by Klejdus et al. [135] using PLE withodified extraction cell content. The modification in the extrac-

ion cell content was made by using 5 mL of a commercial matrixSPE-edTM matrix). For the development of the method, similarxtraction parameters were evaluated, like solvents, number ofxtraction cycles, sample amount, pressure and temperature. Addi-ionally, another innovation of the method was the evaluationf the effect of sonication time before PLE. The extraction yieldramatically increased by using sonication before PLE extractionperformed with 90% MeOH). The amounts of extracted individ-al isoflavones rapidly increased with the sonication time up tomin, and using longer sonication times the increase was lowerednd it was nearly constant after 5 min. However, extraction yield ofglycone (De and Ge) continuously increased with increasing son-cation time until 5 min. The increase in the extraction efficiencyas attributed to the disruption of cell walls by ultrasonic waves.egarding extraction cycles (performed with 90% MeOH), highest

soflavone concentrations in the extracts were obtained after threextraction cycles. However, differences in the yield between twond three cycles were only about 5%.

Most often used solvents (i.e. MeCN, EtOH and MeOH (50 or0–90% in water)), were evaluated for the extraction under PLE.he extraction yields obtained for the extraction of Di and Gi witheCN were about 60–80% (depending of the water percentage) of

he yields of the extraction with MeOH (90%). The extraction effi-iency rapidly decreased with the increasing content of MeCN in thease of Di. Extraction yields between 60% and 75% of the amountxtracted with MeOH (90%) were obtained using EtOH with differ-nt amounts of water (60–90%). Highest yields of both isoflavonesere obtained using 90% MeOH, and linear decrease of extrac-

ion yields were obtained with decreasing content of MeOH in thextraction agent.

Using different amounts of sample, the authors observedecreasing yields with the increasing amounts of sample, obtain-

ng similar results as those obtained by Rostagno et al. [109]. Thisnding was attributed to the thicker layer of sample in the extrac-ion cell. In such study, the influence of pressure was also evaluated.he decrease in the sample size from 0.5 to 0.1 increased approxi-ately 40% the amount of Di and Gi extracted. With the increase of

ressure from 13 kPa to 14 kPa of pressure the amount extractedf both isoflavones increased, and no significant difference wasbserved between 14 kPa and 15 kPa. Increasing extraction tem-erature from 70 ◦C to 110 ◦C produced an increase of the extractionield of Di and Gi (15%), and a much more dramatic increase wasbserved between 110 ◦C and 145 ◦C (60%). The authors claimed thatemperature of about 145 ◦C was most suitable for obtaining max-mal efficiency. The optimized method was used for the analysis of

everal isoflavones different soy samples.

However, the greatest concern regarding the proposed methods that the malonyl forms were not measured and stability wasot evaluated. Using as reference the stability study made by Ros-agno et al. [109] where most malonyl isoflavones are not stable

16 M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 2–29

Table 3Developed methods using pressurized liquids for the extraction of soy isoflavones and evaluated parameters.

Sample used forevaluation of themethod

Isoflavones Fixed extraction conditions Evaluated parameters Selected conditions Reference

Solvent:EtOH (30–80%)MeOH (30–80%)

Extraction cell: 11 mL WaterFreeze-dried soybeans Di, Gi, Gly and

MGiInert material: sea sand Temperature: 60 and 200 ◦C 0.1 g, 100 ◦C, 70% EtOH,

3× 7 min static cycles(∼22 mL)

[109]

Pressure: 100–200 atmSample amount: 0.5–0.05 gStatic cycle length: 5–10 minNumber of static cycles: 1–3 (7 min)and 1–2 (10 min)

Sample amount: 0.2 g Solvent:Extraction cell: 10 mL CH3CNCell content: 5 mL of a commercialmatrix (SPE-edTM matrix)

EtOH (50–90%)

Soy bits Di, Gi, De andGe

Static cycle length: 5 min MeOH (50–90%) 1 min sonication time,0.1 g, 90% MeOH,14 kPa, 2 static cycles(∼20 mL)

[135]

Pressure: 13–15 kPaSonication time: 1–5 minNumber of static cycles: 1–3

Sample amount: 0.5 g Solvent:Extraction cell: 11 mL 58% CH3CN, 58% CH3CN + 5% DMSOInert material: Ottawa sand 70% EtOH, 70% EtOH + 5% DMSO

Soybeans Di, Gi, Gly, De,Ge, Gle, MDi,MGi, MGly, ADi,AGi and AGly

Pressure: 1000 psi 90% MeOH 70% EtOH + 5% DMSO [134]

Temperature: 100 ◦C WaterStatic cycle length: 5 min 95% GenapolNumber of static cycles: 3

Sample amount: 2 g Solvent: MeOH, ACE, CH3CNSoybean flour De and Ge Temperature: 100 ◦C Pressure: 5–15 MPa CH3CN, 2 static cycles,

of 15 min[129]

Inert material: quartz wool/glassbeds

Number of static cycles: n.e.

Extraction time: n.e.

Sample amount: 180 g Temperature: 60–130 ◦CDefatted soybean

flakesDi, Gi, Gly, Deand Ge

Extraction cell: 2 L Pressure: 300–735 psig 110 ◦C, 641 psig, 2.3 h [139]

Solvent: 1800 mL of water Extraction time: 1–3 h

Temperature: 333–393 KPressure: 413–4410 kPa

Defatted soybeanflakes

Di, Gi, Gly, Deand Ge

Extraction cell: 2 L Solvent flow rate: 10–25 mL/min 80% EtOH, 383 K,551 kPa, 25 mL/min,80 g

[140]

Solvent: EtOH:water ratio (0–95%)

D Di: mA aceto

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e: daidzein, Ge: genistein, Gle: glycitein, Di: daidzin, Gi: genistin, Gly: glycitin, MGi: acetyl genistin, AGly: acetyl glycitin, MeOH: methanol, EtOH: ethanol, CH3CN:

nder PLE above 100 ◦C (using 70% EtOH), and a similar dramaticncrease on the yield of glucosides was observed at 150 ◦C, partf the effect of increasing the temperature in the increase of thextraction yield of glucosides may be attributed to degradation ofalonyl isoflavones. Corroborating evidence is that yield of gluco-

ide decreased when extractions were performed above 145 ◦C, aseported by Rostagno et al. [109]. Therefore, the proposed methoday not be able to extract all isoflavones and more importantly,

ithout changing the isoflavone profile of the sample. The sameethod with slight modification (i.e. sonication time of 5 min

nstead of 1 min) was used for evaluation of isoflavone aglyconend glucoside distribution in soy plants and soybeans by Klejdus etl. [136].

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Sample amount: 80–450 g

alonyl daidzin, MGi: malonyl genistin, MGly: malonyl glycitin, ADi: acetyl daidzin,nitrile, n.e: not specified.

Also, Klejdus et al. [137] improved the previous method and usedtwo phase PLE extraction program combined with UAE to extract

soflavones from soy bits. In the first PLE phase, the sample wasxtracted with 2 cycles of 5 min each with hexane at 145 ◦C using45 bar of pressure, followed by a second phase of 2 cycles of 5 minach with 90% MeOH at 145 ◦C using 145 bar of pressure. This is annteresting approach since it allows “cleaning” the sample and per-orming the extraction of target compounds without manipulation

f sample, avoiding the associated errors. However, the same sta-ility issues of the original method [136] persist in the improvedethod.Later, Luthria et al. [134] using the method developed by

ostagno et al. [109], compared several extraction solvents (58%

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eCN, 70% EtOH, 90% MeOH, Water and 95% Genapol) andvaluated the influence of the addition of 5% DMSO to thextraction solvent for the extraction of isoflavones from soy-eans. They observed great differences between assayed solvent.oth, the total isoflavone content and the isoflavone HPLC profilearied significantly with different extraction solvents, achievingighest total isoflavone recoveries from soybean samples withMSO:EtOH:water. 58% ACN extracted only 30.5% of the isoflavonesxtracted with DMSO:EtOH:water. With the addition of DMSO to8% ACN improved extraction to 52.3%. The addition of DMSO to0% EtOH also improved extraction efficiency, while 90% MeOHchieved intermediate yields (83.7%). Very low efficiency wasbtained with genapol or water (18.2% and 13.7%, respectively).

However, since extraction conditions used were optimized byostagno et al. [109] for 70% EtOH, it was expected that the maxi-um efficiency was obtained using DMSO:EtOH:water (5:70:25)

nd EtOH:water (70:30). On the other hand, useful informations provided by the improvement of extraction by DMSO. A possi-le explanation to the improvement of the extraction efficiencyas attributed to the solubility of isoflavones in DMSO reported

y Sigma–Aldrich web site (http://www.sigma-aldrich.com). Theuthors also observed important differences among assayed sol-ents. 90% MeOH extracted the highest amount of glucosides (Gi,i and Gly) and while for the other nine isoflavones the best

olvent was DMSO:EtOH:water (5:70:25). This may explain theesults obtained by Klejdus et al. [135], achieving best yieldsith MeOH than with EtOH, since only glucosides and aglyconesere quantified. An interesting observation was the detection

f all 12 isoflavones by only two extraction solvent mixturesDMSO:EtOH:water (5:70:25) and (DMSO:MeCN:water (5:70:25)).e and MGly were not extracted at detectable levels by the other

olvents. Similarly, Klejdus et al. [136] extracted only trace amountsf De (1.2% of total isoflavones) and did not detect MGly when using0% MeOH.

In contrast, Bajer et al. [129] observed that out of three solventsested (MeOH, MeCN and ACE) MeCN gave the highest yields at00 ◦C. However, extraction was evaluated for some aglycones (Dend Ge only) and the use of a certain amount of water in the extrac-ion solvent was very likely to have influenced the results obtained.fter the pressure was optimized in the range of 5–15 MPa, theumber of cycles and extraction time were also optimized usingeCN. Unfortunately, data regarding the method optimization and

f the influence of the extracting variables were not given.With a different optimization strategy, Li-Hsun et al. [139] used a

teepest ascent design to examine the effect of several independentariables (temperature, pressure and duration) on the extraction ofsoflavones from defatted soybean flakes by superheated water atlevated pressures. They observed that temperature has a greatermpact than pressure and then time, in the extraction of isoflavonessing water. The experimental design revealed that the optimalondition for the extraction of isoflavones was 110 ◦C and 641 psig4520 kPa) for 2.3 h using 180 g of sample and 1800 mL of water.

hen extractions were carried out at higher or lower temperature,r with lower pressure, the total amount of isoflavones decreased.he authors concluded that the decreasing dielectric constant (ε)f water at elevated temperature and pressure might play anmportant role for the enhanced extraction of isoflavones. Thiss indication that the dramatic changes in the physical–chemicalroperties of water, especially in its dielectric constant, at elevatedemperatures and pressures enhance its usefulness as extraction

olvent.

The low extraction efficiency of water observed by Rostagno etl. [109] when compared to the above results can be explained byhe use of a lower temperature (60 ◦C), which is not high enough tohange the dielectric constant of water and increase its effective-

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togr. A 1216 (2009) 2–29 17

ess. An important remark is that not all isoflavone chemical formsere quantified. Also, the large sample amount and solvent volume

nd the inability to extract some isoflavones limit its usefulness asn analytical method. However, the reported results provides anmportant evidence that isoflavones can be extracted using pres-urized water if conditions are optimized enough which could bexploited in future investigations.

Following the same direction, Chang and Chang [140] examinedhe effect of pressure, temperature, solvent flow rate, EtOH:wateratio, and the feed loading on the PLE of isoflavones from defattedoybean flakes using water as solvent. Initially, the effect of solventow and extraction temperature were evaluated and was observedhat using hot pressurized water, increasing the solvent flow ratencreased the extraction efficiency as the extraction time increasedrom 40 min to 200 min. The effect of the increasing temperatureas noticeable between 333 K and 383 K but not between 383 K and93 K (all extraction were performed at 4410 kPa) independently ofhe solvent flow rate (5 and 10 mL/min). These results are similaro those obtained by Li-Hsun et al. [139].

Using pure water or EtOH (at 383 K and 4410 kPa) the laterrocedure extracted more than 50% more isoflavones than water.ecreasing feed loading from 480 g to 180 g increased isoflavonextraction efficiency in approximately 20% after 6 h of extraction,eing in agreement with the trends observed by Rostagno et al.109] and Klejdus et al. [135]. Pressure, however, did not signif-cantly affect the PLE using EtOH with 360 g of feed loading aseported by Rostagno et al. [109]. In contrast, solvent flow have hadn important effect on extraction efficiency, and increasing flowate from 10 mL/min to 25 mL/min increased the total amount ofsoflavones extracted in approximately 15% after 360 min.

The optimization of EtOH:water ratio, feed loading, pressure andolvent flow rate on the recovery of isoflavones of the PLE at 383 Knd 2.3 h was made by means of a four factor Taguchi experimentalesign. EtOH:water ratio and flow rate were the parameters withhe highest influence on the extraction efficiency, while small dif-erences were observed while evaluating feed loading and pressure.n general, the higher the EtOH:water ratio and the flow rate, theigher was the recovery.

Among the variables affecting PLE, the nature of the extrac-ion solvent and temperature generally have profound effects onhe PLE process. When dealing with pressurized solvents, temper-ture will have different impact depending of the solvent usedince physical–chemical properties of each solvent are different.herefore, the best extraction conditions will depend of the sol-ent. The dramatically changes in the physical–chemical propertiesf water, especially in its dielectric constant (ε) at elevated tem-eratures and pressures enhance its usefulness as an extractionolvent. The dielectric constant is a key parameter in determin-ng solute–solvent interactions, and increasing the temperaturender moderate pressure can significantly decrease this constant.t ambient pressure and temperature, water is a polar solvent withdielectric constant (ε = 78) but at 300 ◦C and P = 23 MPa this valueecreases to 21, which is similar to the value of EtOH (ε = 24 at 25 ◦C)r acetone (ε = 20.7 at 25 ◦C). This means that at elevated tempera-ures and moderate pressures the polarity of solvent can be reducedonsiderably and water can be used instead of another organic sol-ent to extract medium-low polarity compounds, or reduce themount of the organic solvent used to achieve effective extractionates [67]. Due to the advantages of lower cost, environmental com-atibility and toxicity, water can be used as extracting solvent if

igh efficiency is not required. However, in view of the available

iterature, pure water, even at elevated temperature and moderateressure, is not as efficient as other solvents for the extraction of

soflavones, despite that the addition of certain amount of water tohe organic solvent is necessary to improve extraction efficiency.

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In general, it is clear that at higher temperatures extraction effi-iency tend to increase, independently of the solvent employed andressure is usually a minor variable (except when using water asolvent) for the resulting efficiency and that it is only required toaintain the solvent in the liquid phase. Another important aspect

f PLE is the stability of isoflavones under extraction conditions.ince some isoflavone are very sensitive to temperature and PLEs performed at elevated temperatures, stability may be the limit-ng factor when using PLE for the extraction without changing thesoflavones profile of the sample. However, this may be considereds positive in the case of hydrolytic methods (see Section 4). To dateo hydrolytic method using PLE has been reported and the poten-ial changes in isoflavone profile using high temperatures can bexplored in future investigations. Also, the influence of the sam-le properties, such as particle size, protein content and enzymaticctivity and its relation with extraction efficiency and isoflavonetability should be investigated in more detail.

.1.2.3. Supercritical fluid extraction. Supercritical fluid extractions the process of separating one component (the extractant) fromnother (the matrix) using supercritical fluids as the extracting sol-ent. A supercritical fluid is any substance at a temperature andressure above its thermodynamic critical point. They can pen-trate samples of plant material almost as well as gases, due toheir high diffusion coefficients and low viscosity. At the same time,heir dissolving power is similar to liquids. Additionally, close tohe critical point, small changes in pressure or temperature resultn large changes in density, allowing many properties to be mod-fied and to obtain selective extraction. The most commonly usedxtracting agent is carbon dioxide (CO2), because of its low cost, lowoxicity, and easily reachable critical parameters (31.1 ◦C/74.8 atm).urthermore, CO2 as a non-polar substance is able of dissolvingon-polar or moderately polar compounds. The addition of a polarodifier (e.g. MeOH) to supercritical CO2 (SC-CO2) is the simplest

nd most effective way to modify the polarity of CO2-based flu-ds in order to increase the solubility of analytes. Modifiers canlso overcome interactions between the analyte and the matrix,ncreasing the extraction efficiency of polar organic compounds113,114,132,141–143].

Although SFE is one of the most complex technique for thextraction of isoflavones due to the high number of possibleariables and interactions, which can effect effectiveness, severalesearchers successfully applied SFE to extract isoflavones from dif-erent soy matrixes such as soy flour, soy hypocotyls and soy cake, asell as from other different matrices, like R. puerariae, M. recutita,. officinalis, F. vulgare and A. eupatoria L. [91,92,118,129,144,145].n overview of the developed methods using supercritical fluids

or the extraction of soy isoflavones and evaluated parameters isresented in Table 4.

As in most modern techniques and methods, stability ofsoflavones under extraction conditions has not been studied so far.his is important since relatively high temperatures are frequentlysed. The same stability principles of the previously discussed tech-iques may apply to SFE and thus it is feasible to consider thathanges in isoflavone profiles can take place during extraction.herefore, evaluation of stability of isoflavones using different SFEonditions, such as temperature, duration and amount and type ofodifier is urgently needed.Regarding the methods developed so far, Chandra et al. [145]

ested a limited number of conditions with different pressures and

mount and type of modifier for the extraction of some isoflavonesDe and Ge) from various soy matrixes. The evaluation of thextraction conditions revealed that at 50 ◦C, 600 atm and 20% EtOHxtracted the highest amount of tested isoflavones (nearly 93%).t is worth noting that the development of the method was per-

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togr. A 1216 (2009) 2–29

ormed spiking reference standards onto a filter paper strip whichas later extracted by SC-CO2. The best-evaluated conditions weresed for the extraction of De and Ge from miso, tofu, and soy mealnd soy flour using sample sizes ranging from 2 g to 10 g. Althoughigh recoveries were achieved, the method was limited in terms of

soflavones quantification.Later, Rostagno et al. [118] evaluated the use of supercriti-

al carbon dioxide for the extraction of soybeans isoflavones (Gi,e and De) using different temperatures, pressures and modi-er concentration. Maximum yield of Gi and Ge was obtained at0 ◦C/200 bar/10 mol%, while maximum yield of De was obtainedt 50 ◦C/360 bar/10 mol%. For the extraction of Gi and Ge, a pre-ominant effect of temperature was observed while for De, aredominant effect of pressure was observed. Also a strong inter-ction between temperature and pressure was observed in thextraction of the tested isoflavones. The decrease in extraction effi-iency with the increase in the temperature can be explained byhe decrease in the supercritical fluid density, while the decrease inxtraction efficiency with the increase in pressure can be attributedo a decreased fluid diffusivity, which may affect interaction withhe sample. However, it is important to note that stability ofsoflavones was not accessed, that only one glucoside (Gi) andglycones were measured and that malonyl glucosides were notuantified. Since relatively high temperatures were used, it is plau-ible that degradation might have taken place during extractionnfluencing the obtained results. The authors suggested that enzy-

atic hydrolysis of Di might have occurred during extraction andnfluenced the results, since the best extraction temperature fore was 50 ◦C, close to the optimal temperature for the activity of-glucosidases.

More recently, Kao et al. [91] modified the experimental con-itions optimized by Rostagno et al. [118] and used 70% EtOHs modifier, instead of 70% MeOH and studied a similar range ofemperature and pressure for the SFE of isoflavones (all 12 mainhemical forms present in soybeans) from soybean cake. The mostmportant aspect of this method, besides the high recovery (87.3%

hen compared to stirring extraction), was the quantification ofll isoflavone chemical forms, since it is the first report of these of supercritical fluids for the extraction of malonyl and acetyl

soflavones. The results showed that a maximum yield of malonyllucoside and glucoside was obtained at 60 ◦C and 350 bar, while aigh level of acetyl glucoside and aglycone was produced at 80 ◦Cnd 350 bar. The highest yield of total isoflavones was obtainedsing 60 ◦C/350 bar, possibly due to predominant concentration ofalonyl and glucosides in the sample. Although a different modifieras used, similar temperature and pressure interaction as reportedy Rostagno et al. [118] was observed.

However, as in most studies, stability was not evaluated andesults might be influenced by degradation of malonyl and gluco-ide isoflavones to their respective acetyl and aglycone forms. Theuthors observed that, although using lower temperatures thanooking and toasting, conversion or degradation can still occurhen in combination with pressure. The amount of malonyl gluco-

ides declined after following a rise in the extraction temperature,hich was suggested to be related with solubility of these chemical

orms or to conversion to acetyl glucoside, glucoside or aglycone,hich may explain highest yields obtained at 80 ◦C.

Araújo et al. [92] also tested different temperatures, pressures,odifiers, and modifier concentration for the SFE of De and Ge

rom soybean hypocotyls after hydrolysis. The highest yields of

hese isoflavones were obtained at 60 ◦C, 380 bar using 3 cycles oftatic and dynamic extraction of 15 min each with the addition of0 mol% of 80% MeCN. Moreover, it was observed that modifiers andressure variations have significant effects in the extraction effi-iency. No isoflavones were extracted without modifiers and the

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 2–29 19

Table 4Developed methods using supercritical fluids for the extraction of soy isoflavones and evaluated parameters.

Sample used for evaluationof the method

Isoflavones Fixed extraction conditions Evaluated parameters Selected conditions Reference

Standards De and Ge

Extraction cell: n.e. Extraction conditions:

600 atm and 20% EtOH [145]

Temperature: 50 ◦C 400 atm and no modifierFlow rate: 950–1000 mL/min 400 atm and 5% chloroformExtraction time: 60 min 400 atm and 5% MeOHRestrictor temperature: 175 ◦C 600 atm and 20% MeOHRinse solvent: none 600 atm and 20% EtOH

Sample amount: 1 gExtraction cell: 7.0 mL (reduced to5.46 mL)Inert material: glass stickModifier: 70% MeOHStatic cycle length: 10 min Modifier concentration: 0.5

and 10 mol%a50 ◦C/360 bar, 10 mol%(TIS and De)

Freeze-dried soybeans Gi, Ge and De Dynamic cycle length: 20 min Temperature: 40–70 ◦C 70 ◦C/200 bar, 10 mol%(Gi and Ge)

[118]

CO2 flow rate: 1.0 mL/min Pressure: 200–360 barExtraction time: 90 min (3× 30 min)Trap: ODSRinse solvent: 1.5 mL MeOHRinse flow rate: 0.5 mL/min

Sample amount: 1 g

[92]

Extraction cell: 10 mLModifier: 70% EtOHModifier concentration: 10 mol%a Malonyl glucosides,

glucosides and TIS:60 ◦C/350 bar

Soybean cake Di, Gi, Gly, De, Ge,Gle, MDi, ADi, MGi,AGi and MGly

Static cycle length: 10 min Temperature: 50–80 ◦C Acetyl glucosides andaglucones:80 ◦C/350 bar

Dynamic cycle length: 20 min Pressure: 300–400 barCO2 flow rate: 1.0 mL/minExtraction time: 90 min (3× 30 min)Fluxing solvent: 5 mL 50% EtOH

Soybean hypocotyls De and Ge

Sample amount: 0.08 g

60 ◦C/380 bar, 10 mol%80% CH3CN

[93]

Extraction cell: 7.0 mL (reduced to5.46 mL)Inert material: glass stickModifier: 70% MeOH Modifier: MeOH, EtOH and

CH3CNStatic cycle length: 15 min Modifier concentration: 0.5

and 10 mol%a

Dynamic cycle length: 15 min Temperature: 50–70 ◦CCO2 flow rate: 1.5 mL/min Pressure: 176–360 barExtraction time: 90 min (3× 30 min)Rinse solvent: 1.5 mL 80% MeOHRinse flow rate: 0.5 mL/min

Soybean flour De and Ge

Pressure: 15–40 MPa35 MPa, 70 ◦C, 5%MeOH, 30 min, 50 �m

[129]Sample amount: 0.3 g Temperature: 10–100 ◦CInert material: glass beads Extraction time: 10–50 min

Restrictor diameter: 25 and50 �mTemperature: 40–70 ◦CPressure: 30–60 MPa

Sample amount: 100 g Modifier composition:MeOH (60–100%)

Soybean meal Di, Gi, De and Ge Extraction cell: 1 L Modifier concentration: 5.4,7.8 and 10.2 mass%a

40 ◦C, 50 MPa,9.80 kg/h, 80% MeOH at7.8% mass, 20–30mesh, 200 min

[147]

Separator 1: 8 ± 0.3 MPa (40 ◦C) CO2 flow rate:3.92–9.80 kg/h

Separator 2: 6 ± 0.3 MPa (30 ◦C) Sample particle size: 10–60meshExtraction time: 0–200 min

De: daidzein, Ge: genistein, Gle: glycitein, Di: daidzin, Gi: genistin, Gly: glycitin, MDi: malonyl daidzin, MGi: malonyl genistin, MGly: malonyl glycitin, ADi: acetyl daidzin,AGi: acetyl genistin, AGly: acetyl glycitin, TIS: total isoflavones, MeOH: methanol, EtOH: ethanol, CH3CN: acetonitrile, n.e: not specified.

a mol% of the CO2 mass passed through the system during the dynamic extraction.

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redominant effect of the pressure in the amount of these twosoflavones (De and Ge) extracted was attributed to the likelyecrease of the steam pressure and increase in the density of fluidnd a higher kinetics of desorption of the compounds from the sam-le matrix. As pressures increases, desorption is faster and moreolute is available for extraction. They also observed similar trends observed by Rostagno et al. [118], where an enhancement ofhe extraction yield by the increase of pressure was dependentf the temperature with correlation of pressure and temperature.lso, major differences were observed for the assayed modifiers.sing 80% MeOH, 80% EtOH and 80% MeCN as modifier, the rela-

ive amount of aglycones extracted were 9.61%, 11.27% and 25.65%espectively when compared to stirring. It is clear that using theroposed method 80% MeCN is much more effective than 80% EtOHnd 80% MeOH.

Another approach is to previously use SC-CO2 to enrich, orclean”, the sample matrix with isoflavones by removing otheromponents from the matrix, as reported by Yu et al. [146], whereoy hypocotyls were defatted by SC-CO2 and used to producesoflavone enriched soy protein.

Bajer et al. [129] optimized the extraction of De and Ge fromoy flour using different pressures (15–40 MPa), temperatures10–100 ◦C) and extraction times (10–50 min). Optimal conditionsere 35 MPa, 70 ◦C, modifier MeOH (5%, v/v) and 30 min. The

uthors reported clogging using restrictors of both 25 and 50 �mf internal diameter and placing the sample between glass bedsvoided the problem. Unfortunately, data obtained during theethod optimization was not provided, which could have been use-

ul for future investigations on the use of supercritical fluids for thextraction of isoflavones.

One of the latest applications of supercritical fluid for the extrac-ion of isoflavones from soy matrixes was recently published byuo et al. [147]. In this report the influence of several extractionarameters, such as pressure, temperature, flow rate, modifier com-osition and concentration as well as sample particle size, wasvaluated for the extraction of some isoflavones (De, Ge, Gi andi) from soybean meal. They observed that using specific condi-

ions (40 ◦C/50 MPa, 5.88 kg/h, modifier flow rate of 0.6 L/h) higherr lower amounts of water than 20% in MeOH (i.e. 80% MeOH)xtraction yield decreased, especially when using high MeOH con-entrations (i.e. 90–100%). This effect may be related with theolarity of the supercritical CO2 which is excessively changed byigh or low amounts of modifier. However, it does not excludehe possibility that if extraction conditions were different (espe-ially temperature and pressure), the highest yield modifier couldave been achieved using a different modifier composition sincextraction conditions may influence CO2 polarity. The authors alsobserved that using 80% MeOH, higher modifier concentrations (i.e.0.2 mass%) resulted in highest extraction yields, which can also bexplained by the changes in CO2 polarity. Here, extraction condi-ions can also influence the effect of the modifier concentration.owever, these results confirm the observations made by Rostagnot al. [118], who also reported that higher modifier concentration10 mol% of 70% MeOH) resulted in higher extraction efficiency.

In most extraction techniques the increase of temperaturewhich may be limited by stability of some isoflavones) usuallyncreases extraction efficiency due to several factors. This may note the case for SFE, since higher temperatures decreases CO2 den-ity (maintaining pressure constant) and thus solvent power of theuid. This issue has been demonstrated by Rostagno et al. [118] and

eflects the results obtained by Zuo et al. [147], who observed thatncreasing temperature from 40 ◦C to 70 ◦C decreased extractionield. Increased extraction temperature favors several processesuch as mass transfer, solubility and volatility of isoflavones andncrease penetration power of SC-CO2, but the positive influence of

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togr. A 1216 (2009) 2–29

hese factors may not be sufficient to counteract the reduced CO2ensity. Also, the higher temperature may increase the solubility ofther sample components in the SC-CO2 and reduce the extractionfficiency of isoflavones. Pressure (at 40 ◦C) was also evaluated byuo et al. [147] and a straightforward trend was observed, achiev-ng highest yields using higher pressures, possibly due to increasedO2 density. Increasing CO2 flow rate increased yields, obviouslyue to increased mass of CO2 used. In this report, the most interest-

ng extraction parameter evaluated (which has not been assessed soar) was the influence of sample particle size. Reducing particle sizerom 1.19 mm (10–20 mesh) to 0.68 mm (20–30 mesh) improvedxtraction yields and smaller particle size decreased extractionields. Smaller particles provide greater contact surface and betterllow the penetration of the SC-CO2 and consequently extractionfficiency increase. The explanation given by the authors to theecrease of yields observed when using smaller particle sizes than.68 mm was the aggregate formation, which can cause the fluid tohannel or short circuit.

One of the limiting aspects of this study was the use of one-at-ime strategy to optimize extraction conditions, which is not the

ost recommended to be used for supercritical fluids, since therere much more variables and interactions that can more severelynfluence effectiveness than in other extraction techniques. Theest approach when using SC-CO2 seems to use experimentalesigns or, preferably, a full screening of extraction conditions.lso, the quantification of only glucosides and aglycones providenly limited information on the effect of evaluated parametersn the extraction of malonyl and acetyl glucosides, which haveeen demonstrated to be the most difficult isoflavone forms to bextracted by supercritical fluids.

There are a large number of variables that can affect the extrac-ion using SFE, including not only pressure, temperature and typend amount of modifier, but also others such as the duration ofynamic and static cycles, CO2 flow rate, thimble times swept,estrictor temperature, trap composition, rinse solvent and rinseow rate. Trap composition and elution conditions (solvent, flowate and temperature) have not been studied so far and maytrongly influence isoflavone recovery and need to be investigatedn the future. Also the sample is an important factor since the par-icle size and interactions with the matrix (i.e. protein content)

ay greatly influence the ability of the supercritical fluid to extractarget compounds. Other sample characteristics may influence sta-ility during extraction (i.e. glucosidase activity). The presencef other sample components, such as oil, may interfere with themount of target compounds that can be extracted depending ofhe fluid density. Moreover, extraction conditions can have a greatmpact on the effectiveness of a certain extraction parameter, andelection of extraction variables should be carefully studied in ordero achieve maximum effectiveness. Therefore, much research is stilleeded to fully determine the potential of supercritical fluids for thextraction of isoflavones from soybeans and soy foods.

.1.2.4. Microwave-assisted extraction. Microwaves are electro-agnetic waves with wavelengths ranging from 1 mm to 1 m, or

requencies between 300 MHz and 300 GHz. Microwave-assistedxtractions are based on absorption of microwave energy byolecules of polar chemical compounds. The energy absorbed is

roportional to dielectric constant of the medium, resulting in rota-ion of dipoles in an electric field (usually 2.45 GHz). The efficiencyf MAE depends on several factors, such as solvent properties, sam-

le material, the components being extracted, and, specifically, onhe respective dielectric constants. The higher dielectric constant,

ore energy is absorbed by the molecules and the faster the solventeaches the extraction temperature. In most cases, the extract-ng solvent has a high dielectric constant and strongly absorbs

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icrowave radiation. Microwaves may also promote selective andapid localized heating of moisture in the sample by microwaves.ue to the localized heating, pressure builds up within the cellsf the sample, leading to a fast transfer of the compounds fromhe cells into the extracting solvent. Additionally, by using closedessels the extraction can be performed at elevated temperaturesabove boiling point of the solvent) accelerating the mass transferf target compounds from the sample matrix [113,114,132,148].

This technique has been used only recently and on a fewccasions to extract isoflavones from soybeans and from dif-erent matrixes, such as R. astragali, R. puerariae and peanuts86,110,124,149,150]. An overview of the developed methods using

icrowaves for the extraction of soy isoflavones and evaluatedarameters is presented in Table 5.

Since high temperatures are usually used by MAE is conve-ient to discuss the stability of isoflavones during extraction beforeetailing developed methods. There is apparently only one studyf soy isoflavone stability during MAE, which was reported by Ros-agno et al. [110]. They evaluated stability of soy isoflavone extracts50%EtOH) at different temperatures (50–150 ◦C) for 30 min andbserved that temperatures above 50 ◦C significantly changedsoflavone profile of the extracts mainly due to conversions between

alonyl and their respective glucosides. Malonyl isoflavones wereot detected above 100 ◦C and temperatures higher than 125 ◦Cromoted hydrolysis of glucosides to their respective aglycones.

Regarding the developed methods, several extraction solvents,emperatures, and extraction solvent volume, as well as the sam-le size and extraction time were studied by Rostagno et al. [110]or the optimization of an extraction protocol for all main soysoflavones. In the first selection of the most adequate extractionolvent, pure solvents (MeOH, EtOH and water) extracted lowermounts than 50% EtOH and 50% MeOH. Since 50% EtOH extractedhe highest amounts of total isoflavones, it was further studiedn terms of water percentage (30–70%) and results indicated thatsing higher or lower amounts of water than 50% reduced extrac-ion efficiency. A similar result was obtained with solvent volume;sing high or low solvent volumes resulted in lower extraction effi-iency than intermediate volumes (20–30 mL). After solvent andolume were optimized, sample amount was evaluated revealinghat using low sample size reduce extraction efficiency and sam-le size greater than 0.5 g does not improve yield. Therefore, theuthors concluded that a sample:solvent ratio of 0.5:25 (g/mL)esults in maximum efficiency using the optimized conditions soar. The effect of sample size is different when using MAE than withther techniques, such as PLE, which increases extraction efficien-ies using smaller samples. Examining the effect of extraction timehe authors obtained a straightforward response: increasing extrac-ion time increased extraction yield, and quantitative extractionsere obtained in 20 min. They also observed that most isoflavonesresent in the sample (approximately 75%) were extracted in therst 10 min of extraction. No isoflavone degradation was observedsing the developed extraction protocol and a high reproducibilityas achieved (>95%).

The most interesting point of this report was the proposal ofn effectiveness factor in order to evaluate extraction conditions.ost authors simply use total isoflavones as reference of extraction

fficiency. However, sometimes there is no significant difference inhe total isoflavone yield although there are significant differencesetween chemical forms, and selection of a certain solvent can beomplicated procedure. In the case of the proposed effectiveness

actor, it balances the extraction effectiveness for all isoflavonesnd more accurately identify the most adequate solvent.

Also recently, Careri et al. [86] adopted a fractional facto-ial design to develop a hydrolytic method for the extraction ofsoflavonoid aglycones (Di, De, Gi, Ge, Biochanin A and Formonotin)

(ipce

togr. A 1216 (2009) 2–29 21

rom yellow soybeans using microwave-assisted extraction. Forevelopment of the method, several extraction parameters werevaluated such as microwave power, extraction time, solvent,xtraction volume, acid concentration and re-hydratation time.everal interactions among extraction variables were found andhe most relevant parameters resulted to be the microwave power,he extraction time and the acid concentration. It is important toote that sample was submitted to sonication for 15 min beforextraction using MAE, which may have extracted most isoflavonesn the sample, as previously discussed, and the proposed methodan almost be considered a hydrolysis method rather an extractionethod.It is clear that although highly efficient extractions can be

chieved with MAE, its potential is limited since only short extrac-ions can be used due to isoflavones stability. However, this maye very useful when proposing hydrolytic methods, which can ben interesting application for this extraction technique. Also, moreesearch is needed to determine the influence of other parametersot only on extraction but also on stability, such as pressure. Sampleharacteristic such as humidity, �-glucosidase activity, and particleize also need to be investigated in future researches.

.2. Liquid samples

Apart of solid samples, there are liquid soy samples that alsoontain isoflavones. Most common liquid soy foods are soy milk andoy beverages blended with fruit juices. Usually, these samples arereeze-dried and treated as solid samples, using the same methodsnd techniques [41,44,46–48,87,102,151,152].

The problem with the freeze-drying procedure is that it can takeays and may as well, increase variations on the determination of

soflavones, due to increased errors and degradation of the sam-le. Moreover, it goes in the opposite direction of the recent trendf sample preparation that is to use fast methods and reduce to ainimum the necessary steps from sample to analysis. It is not log-

cal to have at hand an extraction method that can be accomplishedn 20 min and use a sample pretreatment of days.

Liquid samples are similar to a solid sample in that mostsoflavones are in the suspended solids or already in the liquidhase and therefore an extraction step can be used to extract the

soflavones from the solids and avoid freeze-drying the sample.ndeed some authors successfully direct extraction of liquid sam-les without freeze-drying. Most authors used methanol (MeOH)r ethanol (EtOH) with a sample:solvent ratio ranging from 4:1o 1.6:1 (v/v) and extraction by refluxing or shaking for 1–4 h51,77,153,154].

These methods were adapted from protocols used for solid sam-les and more importantly, were not evaluated, very long extractionimes were used or only a few isoflavones were studied. Also, these of refluxing causes malonyl isoflavones to undergo degradationo the respective glucosides and aglycones, changing the isoflavonerofile of the samples and limiting the information obtained. On thether hand, they indicate that an extraction step can used instead ofreeze-drying the sample. This was demonstrated by Rostagno et al.151], who recently developed a new method for the fast determi-ation of isoflavones from soy beverages blended with fruit juicessing UAE.

During the method development, several parameters weretudied such as solvent (methanol and ethanol), sample:solventatio (5:1 to 0.2:1), temperature (10–60 ◦C) and extraction time

5–30 min). The most important parameter for the extraction ofsoflavones from soy drinks was the sample:solvent ratio. Also, sam-les were freeze-dried, extracted using a reference method andompared with the optimized method and no significant differ-nce was observed on total and individual isoflavone concentration.

22 M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 2–29

Table 5Developed methods using microwaves for the extraction of soy isoflavones and evaluated parameters.

Sample used for evaluationof the method

Isoflavones Fixed extractionconditions

Evaluated parameters Selected conditions Reference

Freeze-dried soybeansDi, Gi, Gly, De, Ge, Gle,MDi, MGi, MGly, ADi,AGi and AGly

Solvent:WaterEtOH, 50% EtOH

Solvent: 25 mL MeOH, 50% MeOHMicrowave power:500 W

EtOH (30–70%) 0.5 g, 50 ◦C, 20 min and50% EtOH

[110]

Magnetic stirring: 50%nominal power

Temperature: 50 and 150 ◦C

Solvent volume: 15–35 mLSample amount: 0.1–5.0 gExtraction time: 10–30 min

Soybeans Di, De, Gi, Ge, BiochaninA and Formononetin

Microwave power: 10 and 600 W600 W, 1 min, 3 mL of80% CH3CN, 12 M HCland no re-hydratationtime

[87]

Extraction time: 1 and 8 minSample amount: 0.1 g Solvent: 80% CH3CN and 80% MeOHSonication beforeextraction: 15 min

Solvent volume: 3 and 8 mL

Acid concentration: 1 and 12 M

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he novelty of this work resides in its simplicity and rapidity whenreating a troublesome liquid sample without the need of freeze-rying the sample before extraction. This report provides valuable

nformation although further evaluation of the influence of otherxtraction parameters, such as sample characteristics, ultrasoundrequency and power and the use of ultrasonic pulses is still needednd will likely be explored in future investigations.

Another sample preparation technique that can be used forxtracting soy isoflavones from liquid foods is solid phase extrac-ion. SPE involves adsorption of sample components on the surfacef a solid sorbent, followed by elution with a selected solvent. Aariety of sorbents available in the market allows not only the iso-ation of analytes, but also the removal of interferences. However,he whole potential of this technique for the analysis of isoflavonesn foods is yet to be determined. Although SPE applications for thenalysis of isoflavones from blood, plasma, urine and serum areelatively common [155–161], only a few works explored the SPEotential for the analysis of isoflavones from liquid samples.

Mitani et al. [162] for instance, proposed an automated on-linen-tube solid phase microextraction (SPME) coupled to HPLC forhe determination of daidzein and genistein in soy foods. In-tubePME is a preconcentration technique using an open tubular fused-ilica capillary with an inner surface coating as the SPME device,hich can be easily coupled on-line with HPLC. In tube SPME allows

or convenient automation of the extraction process, which notnly shortens the analysis time, but also provides better accuracy,recision and sensitivity relative to off-line manual techniques.owever, a hydrolysis step was required because the isoflavone glu-osides present in the sample were difficult to concentrate usinguch conditions, which limit its usefulness for quantification of allsoflavone chemical forms. Moreover, since most isoflavones aren the suspended solids in liquid samples, it is very likely that anxtraction step before the in-tube SPME method will be required;therwise it will only separate isoflavones present in the liquidhase and may lead to an underestimation of isoflavone concen-rations.

Therefore, the greatest potential of this technique is the concen-

ration and clean-up of extracts coupled to an extraction techniqueuch as PLE or SFE, or after the extraction procedure with one of thereviously discussed methods. It also may be used before extractiono eliminate undesirable components of the sample and allowing a

ore selective extraction of target compounds.

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Re-hydratation time: 0 and 10 h

alonyl daidzin, MGi: malonyl genistin, MGly: malonyl glycitin, ADi: acetyl daidzin,nitrile.

.3. Optimization of extraction conditions

In view of the fact that chemical modifications of isoflavonesay occur during the extraction process, not only isoflavone extrac-

ion efficiency for a particular solvent need to be considered whenomparing the extraction solvents, but also preservation of origi-al isoflavone composition during extraction, minimizing chemicalransformations.

Concentrations of �-glucosides and acetyl glucosides formsould be increased or decreased during extraction procedure, andglycones could be increased as a consequence of chemical trans-ormations. Higher amounts obtained of these chemical forms doot necessarily mean higher extraction efficiency since it could beesult of the transformations. Thus, it is difficult to determine theost efficient solvent for extracting �-glucosides, acetyl glucosides

r aglycone isoflavones by simple comparison of yields. Malonylsoflavones are the chemical forms most susceptible to degradationnd therefore the higher amount of malonyl glucosides present inhe extracted material indicates either higher extraction efficiencyf the solvent, better protection from chemical transformations oroth. In the case of not quantifying malonyl isoflavones the besthoice is to determine the stability during extraction.

Apart of stability issues, extraction conditions should beptimized for each solvent and for each sample. Quantitativeecoveries can be achieved with most commonly used solvents forhe extraction of isoflavones from soybeans and soy foods, givenxtraction conditions are optimized enough. Therefore, the dis-ussion of which is the best solvent should be addressed in termsf advantages and disadvantages. For example: MeCN sometimesan extract more isoflavones than MeOH or EtOH. However, MeCNave a higher cost, toxicity and lower environmental compatibilityhan EtOH and MeOH and if quantitative recoveries are achievedith these solvents there is no justification for the use of MeCN

s extractant. Also, the influence of the sample should not beverlooked. It can seriously affect the ability of a given solvento extract some isoflavones and use a method optimized with aertain sample should not be used without evaluation even for a

imilar sample. An internal standard can be used to account forosses during extraction for quantification studies. An ideal internaltandard should be a compound structurally related to the analytend with a similar polarity, but with a retention time that doesot overlap the other peaks during the chromatographic analysis.

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piking the sample may also be used to estimate if quantitativextractions are achieved, but enough time should be given for thetandards interact with the sample. However, interaction with theample matrix cannot be ensured even if prolonged soaking issed. Also, it may be of interest to use an extract obtained withptimized conditions rather than pure standards, since an extractore closely resemble the sample. Finally, sequential extractions

re seriously recommended to ensure that quantitative recoveriesre achieved [106,107,109,151].

Overall, to optimize extraction conditions three strategies cane used: one-variable-at–a-time optimization, the use of an exper-

mental design, or a combination of both. The one at a timepproach is more objective and can be used to isolate the effectf a given variable and provide easier interpretation. The draw-ack of this strategy is that it is somewhat limited to observe

nteractions among extraction variables. For instance, when opti-izing an extraction using PLE, MeOH extracted higher amounts

f isoflavones than EtOH at 60 ◦C and 100 bar of pressure. However,f higher temperature or pressure was used, the physical–chemicalroperties of the solvents change and MeOH may not obtain higherields than EtOH. To observe interactions, either a screening of allossible extraction conditions or an experimental design can besed. Screening implies a huge number of extractions, which takesime and have high cost. The experimental design allows reduc-ng the number of analysis needed to identify the most importantxtraction variables. An excellent optimization strategy would beo use an experimental design to identify the most important vari-bles, and further investigate these variables in detail by screening.lso, the use of mathematical models and computer aided opti-ization may be valuable tools for reducing the time required to

evelop extraction methods hampered by the existence of a greatumber of variables influencing the process and will likely explored

n the future.

.4. Critical comparison of extraction methods

Serious efforts have been made in the last decade trying toompare techniques, methods and solvents for the extraction ofoy isoflavones. A relative comparison of extraction techniques/ethods available in the literature is shown in Table 6.Ascertaining the most suitable extraction technique/method/

olvent for determination of isoflavones in soy samples is rela-ively difficult. In spite of the fact that the substances investigatedre quite close chemically, physically and physiologically, there aremportant differences on the extractability of each chemical formnd isoflavone type, and therefore it is almost impossible to sug-est a single extraction solvent that ensures that all isoflavonoidsre extracted with maximum yields from all types of soy samples107,129]. It is also important to remark that, in most cases, whatre being compared are extraction methods using different extrac-ion techniques and not the extraction technique itself. Stability

ay also affect results and provide incorrect relative comparisonalues.

Rostagno et al. [118], for example, developed an extractionethod for the extraction of some isoflavones from soybean

our using SFE and compared the results obtained with differ-nt techniques (UAE and soxhlet). Highest yield of total isoflavonesvaluated were obtained by UAE, followed by soxhlet and SFE meth-ds. Soxhlet extracted 70%, and SFE extracted approximately 30%f the amount obtained by the UAE method, respectively. How-

ver, there are several factors that may responsible for the observedifferences among extraction methods that may be attributed tohe extraction method rather than to the technique itself. In thisase, the most important is that stability was not evaluated andnly a few isoflavone chemical forms were quantified. This is of

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togr. A 1216 (2009) 2–29 23

ttermost importance since using soxhlet extraction, degradationf isoflavone malonyl, acetyl and glucoside forms is known to takelace increasing concentration of glucosides and aglycones andherefore results may not be comparable. Also, performing UAEor 90 min is very likely to increase extraction temperature andn similar basis and promote degradation of isoflavone conjugateorms. Therefore, these results should be taken with caution andhe relative efficiency of SFE may be underestimated.

The comparison of extraction methods can be very relative,specially regarding the method used as reference. Araújo et al.92] compared SFE and stirring with for the extraction of somesoflavones from soybean hypocotyls. The proposed SFE methodxtracted 25.65% of the aglycones extracted by stirring. The relativemount extracted reported is overestimated since the hydrolysisas only applied to samples extracted by the SFE technique and not

o the reference method (stirring). Hydrolysis markedly increaseshe concentration of aglycones present in the sample at the expensef the glucosides and hence increases the available amount of agly-ones to be extracted and the actual yield may be much lower. Also,he necessary corrections when using hydrolytic methods were notpplied which further influence results obtained.

The authors go even further and claim the results obtained withhe proposed method were superior to those obtained by Chan-ra et al. [145] and Rostagno et al. [118]. The yield of aglyconesbtained by Araújo et al. [92] was 180 �g/g while Chandra et al.145] extracted between 15 �g/g and 103 �g/g dry weight (depend-ng of the sample) and Rostagno et al. [118] extracted 32.6 �g/g.

key point for the differences between these reports is also theydrolysis of the extracts used by Araújo et al. [92]. Comparingields of only aglycones between hydrolytic and non-hydrolyticethods is rather complicated and even more is the non-hydrolyticethods used as reference quantify only a few glucosides [118] or

o glucosides at all [145]. If all chemical forms are quantified its possible to compare hydrolytic and non-hydrolytic methods by

aking the corrections for the molecular mass. Moreover, differentamples and sample types were used. Araujo et al. [92], extractedsoflavones from soy hypocotyls, Chandra et al. [145] from miso,ofu, soy meal and soy flour and Rostagno et al. [118], from soyour. The concentration of a given compound on the sample directlyffects the yields and comparison of different samples should beased on relative recoveries rather than in yield. Also, sample stabil-

ty may be affecting the recoveries of the earlier reports, thus theseesults may not be comparable in the same basis. Therefore, authorshould be very careful when making assumptions when compar-ng results with those obtained with other methods available in theiterature.

Kao et al. [91], compared SFE and shaking for the extraction ofsoflavones from soybean cake and observed that shaking extractedigher amounts of total isoflavones (approximately 35%) than theighest amounts obtained with SFE (60 ◦C/350 bar). However, SFExtracted higher amounts of acetyl glucosides and aglycones. At0 ◦C/350 bar SFE extracted approximately 33% and 91% of mal-nyl and glucosides, respectively than the amount obtained withhaking, while shaking extracted 80% and 87% of the acetyl andglycones, extracted by SFE. At 80 ◦C/350 bar, SFE extracted evenesser malonyl and glucosides (17% and 70%, respectively) thanhaking, and shaking extracted even less acetyl and aglycones65% and 58%, respectively) than SFE. These observations may haveeen influenced by degradation of malonyl isoflavones and withoutroper evaluation of isoflavone stability results might give a false

mpression of effectiveness.Also, the relative yield obtained with the developed method

65%) is much higher than those obtained by Rostagno et al. [118]28%) and Araújo et al. [92] (26%). However, the relative yieldseported by Rostagno et al. [118] (28%) and Araújo et al. [92] were

24 M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 2–29

Table 6Relative comparison of extraction techniques/methods.

Sample Isoflavones Compared techniques/methods Relative yield (%)a Reference

Soy flour Gi, Ge and De SFE/UAE/Soxhlet 28/100/68 [118]Soybean cake Di, Gi, Gly, De, Ge, Gle, MDi, MGi,

MGly, ADi, AGi and AGlySFE/Shaking 74/100 [92]

Soybean hypocotyls De and Ge SFE/Stirring 26/100 [93]Soybean meal Di, Gi, De and Ge SFE/Stirring 87/100 [147]Soybeans Di, Gi, Gly and MGi UAE/Stirring 100/85–100b [106]Soybeans Di, Gi, Gly, De, Ge, Gle, MDi, MGi,

MGly, ADi, AGi and AGlyPLE/UAE/Soxhlet/Shaker/Vortex/Stirring 100/93/68/71/66/70 [134]

Soy bits Di, Gi, De and Ge PLE/UAE/Soxhlet/PLE + UAE 49/14/64/100 [135]Soy bits Di, Gi, Gly, Ononin, De, Gle and Ge UAE/Soxhlet/PLE + UAE 22/68/100 [137]Soy flour, Meat substitute,

nuts and protein isolateGi, MGi, AGi and Ge PLE/Stirring 98–100/88–100c [133]

Soy flour De and Ge UAE/UHOM/SFE/PLE/Soxhlet 100/93/16/71/69 [129]Soy flour Di, Gi, Gly, De, Ge, Gle, MDi, MGi,

MGly, ADi, AGi and AGlyMAE/UAE 100/100 [110]

De: daidzein, Ge: genistein, Gle: glycitein, Di: daidzin, Gi: genistin, Gly: glycitin, MDi: malonyl daidzin, MGi: malonyl genistin, MGly: malonyl glycitin, ADi: acetyl daidzin,AGi: acetyl genistin, AGly: acetyl glycitin, UHOM, Ultrasonic homogenizer.

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a Relative to the technique which extracted the highest amount of total isoflavonb Depending of the solvent used.c Depending of the sample used.

nfluenced by degradation during extraction with the referenceethod and by the use of hydrolytic methods. Since stability was

ot accessed by Kao et al. [91] the relative yield, although muchigher than those reported earlier, is only speculative.

Zuo et al. [147], achieved even higher relative recoveries (87%)han Kao et al. [91] when compared to solvent extraction using

agnetic stirring using different extraction conditions. In this case,nly a few isoflavones were quantified (glucosides and aglycones)nd results were reported in only in terms of total isoflavones andhe issues of the previous discussed method apply.

Stability during extraction may be one of the most importantspects when comparing extraction techniques/methods since theyay change isoflavone profile of the sample and affect yields, which

s especially important when all isoflavone chemical forms are notuantified. For the reliable comparison of extraction techniques,xtraction conditions should be optimized for each technique,nsure that using the optimized conditions do not affect isoflavonerofile and only then, results might be comparable. One option tobtain comparable results between methods/techniques is the usef hydrolytic methods, including the reference method, since it willliminate variations derived from transformations. The handicapf using hydrolysis is that only limited information is obtained (i.e.otal isoflavones) although it may prove useful in some cases.

Several other authors tried to compare different extraction tech-iques/methods. Rostagno et al. [106] compared UAE and magnetictirring using several different solvents (water, EtOH, MeOH andeCN with different water percentages) at 10 ◦C for 10 min and

bserved that UAE extracted between 0% and 15% more isoflavoneshan magnetic stirring at 10 ◦C, depending of the solvent. At 60 ◦Cimilar increase in extraction efficiency was observed. Also, a sim-lar solvent response was observed using magnetic stirring andAE, achieving maximum extraction efficiency using solvents with0–60% water.

Luthria et al. [134] compared several extraction techniques (stir-ing, shaker, UAE, vortexing, soxhlet and PLE) of 12 main isoflavonesrom soybeans using the same solvent (DMSO:EtOH:water5:70:25) as extracting solvent. PLE was the most effective methodor the extraction of total isoflavones, extracting between 30% and

5% more isoflavones than the other methods. Total isoflavonesxtracted by UAE was 93.3% as compared to PLE and shakingxtracted 75.6% of amount extracted by UAE. Both, the totalsoflavone content and the isoflavone HPLC profile varied signifi-antly with different techniques. MGly and De were detected only

a

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n PLE and UAE extracts. Shaking and stirring extracted the highestmounts of malonyl isoflavones (MDi and MGi) while PLE extractedhe highest amount of acetyl glucosides. Extraction conditionssample size, extraction length, number of extraction cycles andemperature) used in the PLE procedure were point by point opti-

ized by Rostagno et al. [109] while the other extraction methodsere not, and therefore is not surprising that PLE revealed to be theost effective extraction technique.Klejdus et al. [135] evaluated different techniques/methods (PLE,

AE, soxhlet and PLE + UAE) for the extraction of isoflavones (De,e, Di and Gi) from soybean foods. PLE + UAE (1 min sonication)xtracted the highest amount of isoflavones followed by soxhlet,LE and UAE, in this order. Soxhlet extracted the highest amountf aglycones while PLE + UAE extracted the highest amount of glu-osides. However, malonyl isoflavones were not quantified andtability was not accessed and the highest amount of isoflavonesxtracted by PLE, PLE + UAE and soxhlet than by UAE alone may beartially attributed to degradation of malonyl isoflavones leadingo their respective glucoside and aglycone forms.

Later, Klejdus et al. [137] evaluated soxhlet, UAE and PLE + UAEor the extraction of isoflavones from soy bits and obtained similaresults. In this case, however, sonication time before PLE was 5 minnstead of 1 min. The same stability issues of the later report alsopply here.

Downing et al. [133], compared PLE and the method developedy Barnes et al. [78] using stirring. The method by Barnes et al.78] required 60 min, while the PLE procedure (performed at 80 ◦C)equired 20 min to extract similar levels of genistein equivalents.hey observed significant differences on the extraction of conju-ated forms of genistein extracted by these two methods. Heaturing PLE caused significantly less acetyl genistin to be present

n the extracts when compared with stirring where ambient tem-erature was used. However, this outcome was dependent of theample. In some soy flour samples deesterification occurred and inthers not. Acetyl genistin was much more susceptible to degra-ation than malonyl genistin and degradation of the former onlyccurred in one sample (soy nuts). The change in the forms ofenistein was attributed to heat-induced deesterification of the

cetylgenistin and malonyl genistin to genistin.

Bajer et al. [129] also evaluated different extraction methodsUAE, ultrasonic homogenizer, SFE, PLE and soxhlet) for the extrac-ion of De and Ge from soy flour using optimized conditions. Theybserved that the different isoflavones present in the assayed

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amples are extracted in maximum yields by different methods.he highest amount of these two isoflavones was obtained by UAE,ollowed by ultrasonic homogenizer, PLE, soxhlet and SFE, in thisrder. No significant difference was observed between PLE andoxhlet. For De, the best extraction methods were UAE and soxhletollowed by ultrasonic homogenizer, PLE and SFE, in this order,hile for Ge the extraction methods with highest yields wereltrasonic homogenizer, UAE, PLE, soxhlet and SFE, in this order.owever, these results should be taken with care. Stability wasot assessed nor was the malonyl and glucoside forms quantified.oxhlet extraction is known to promote degradation of these formsnd nevertheless, extracted low amounts of Ge (more than half ofhe amount extracted by ultrasonic homogenizer). Moreover, noata on the optimization of each extraction method was providednd therefore these may be rather speculative.

There is a fundamental difference when comparing the extrac-ion of a given compound, comparing the extraction techniqueUAE, PLE, etc.) and the extraction method. Using the same tech-ique is possible to have two different quantitative extractionethods. In most cases, authors compare different extraction tech-

iques using different extraction methods and therefore to drawonclusions from this kind of reports is difficult. If the aim of thetudy is to optimize an extraction method using a certain techniquend make a comparison with other methods (that use differentxtraction techniques), it is essential that authors, use optimizedonditions for all extraction methods and ensure that they do notffect isoflavone profile of the sample. Comparison with a referenceethod reported in the literature may also be used.An illustrative example for the comparison of the different

xtraction methods/techniques can be taken from the work of Ros-agno et al. [110]. These authors proposed an MAE method afterptimizing several extraction conditions and evaluated isoflavonetability with the optimized method, which did not affect isoflavonerofile in the sample. The MAE method was compared with a previ-us developed method using UAE and no difference was observed inotal and individual isoflavone yields. With both methods, quanti-ative extractions were obtained in 20 min. In this case, not only arehe methods comparable but also the extraction technique, whichre similarly effective for the extraction of isoflavones from soy-eans. Moreover, quantitative recoveries are achieved with bothechniques without changing the isoflavone profile of the sample.

Altogether, SFE seems to be less efficient for extraction ofsoflavones than other techniques, while UAE, PLE and MAE, the

ost efficient. Apart from extraction efficiency, there are otherspects that are important when determining the most suitablextraction technique. Selection of an appropriate extraction tech-ique entails consideration of not only the recovery but also theost, time of extraction, and the volume of solvent used, amongthers. From the point of view of solvent consumption, SFE is with-ut doubt the best extraction technique. In contrast, soxhlet requirearge amounts of solvent and is a time-consuming procedure and

ay affect isoflavone profile. UAE and MAE have demonstrated toe fast techniques for the extraction of isoflavones from soybeans,ollowed closely by PLE. SFE is an intermediate option. Also, theeed of post-extraction purification steps is an important issue. PLEnd SFE provide sufficiently pure extracts without the need of sub-equent filtration, while using UAE the filtration step is required.LE has been shown to have important advantages over compet-ng techniques as regards time saving, solvent use, automation andfficiency. PLE and SFE have the advantage that no filtration step is

eeded, since the matrix components that are not dissolved in thextraction solvent may be retained inside the sample cell. Also, itllows to easily performing reextractions of the sample to ensureuantitative extractions are achieved. PLE and SFE are very conve-ient for the purposes of automation and on-line coupling of the

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togr. A 1216 (2009) 2–29 25

xtraction and separation techniques. Also, PLE and SFE offers theossibility of performing the extractions under an inert atmospherend protected from light, which represents an attractive advantageince many compounds, are sensitive to these two external factors67,109]. However, some modern extraction techniques (MAE, PLEnd SFE) are not always available in the average laboratory, due tohe high cost of the equipment.

For the analysis of a particular sample with approximate knowl-dge of concentration and distribution of isoflavones, such as foroutine quality control of similar soy flours, UAE can be used dueo its low cost and high efficiency. In contrast, for the analysisf different samples with unknown isoflavone concentration andistribution, PLE may be preferred since besides high efficiency

t allows to easily perform reextractions of the sample. Thus, thehoice of an extraction technique will depend of several factorsesides efficiency. Among these factors, implicit characteristic ofhe techniques are particularly relevant such as instrumental cost,evel of automation and possibility of on-line coupling with analysisechnique. A good way to select an appropriate extraction techniques to consider practical aspects and establish a multicriteria decision

aking procedure using desirability function optimization.

. Post-treatment of extracts

After extraction of isoflavones from the sample matrix is per-ormed, the extract can be submitted to a series of post-treatmentteps before the analysis. These procedures can be reduced to ainimal depending of extraction technique used. After extraction,

nsoluble materials are usually removed by filtration or centrifuga-ion and sometimes, the extract are immediately analyzed withouturther preparation. If extract is obtained using PLE or SFE filtra-ion and centrifugation is not required. Also, several authors simplyass the sample through 0.45 �m filters after extraction and avoidhe centrifugation step [86,91,109,110,134,144,147,151]. The limita-ion of not using centrifugation is the difficulty of correcting theample volume and solvent losses during filtration, especially withmall samples. This problem can be prevented by using an inter-al standard with the specific aim of correcting the sample volume81,106,109,110,151].

After filtration, liquid–liquid extraction can be used to removendesired sample components such as the lipophilic components,

n order to preserve reverse phase chromatographic columns.ydrolysis of the extracts can also be used after the extraction using

he same methods discussed in Section 4.Another common post-extraction procedure is the partial or

omplete removal of the solvent by rotary evaporation and re-issolution of the sample either on the mobile phase used for thehromatographic analysis or in 80% MeOH [41,42,72,90,102,107,129,33,135–137,144,152].

This procedure can be used to pre-concentrate the extracts andeduce detection and quantification levels during chromatographicnalysis. Another reason for this post-extraction step is to avoidhe peak distortion caused by injecting samples containing highoncentration of MeCN onto columns equilibrated with low MeCNoncentration. The procedure is time-consuming and such han-ling always increase variability and can be source of losses andegradation. Moreover, the use of this post-extraction step can bevoided by using a compatible solvent for extraction such as EtOHr MeOH, by limiting the sample size to less than 5 �L when usingonventional C18 columns or by using high flow HPLC methods with

onolithic columns [81,104]. Avoiding the use of such cumber-

ome procedure can greatly decrease the time required for samplereparation.

More often, some additional sample preparation are used to iso-ate analytes of interest from other sample components that can

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nterfere with the chromatographic analysis or as an extract enrich-ent step, wherein the analyte concentration is increase above

he determination limit of the final determination technique. SPEs one of the most used enrichment techniques. SFE have beensed by some authors [52,129,163,164] to provide a clean con-entrated isoflavone extract to be used in the chromatographicnalysis.

A wide selection of sorbents, ranging from classical C8 and C18ilica based sorbents to new polymeric materials, enables substan-ial selectivity of the enrichment process. Most traditional solidhase extraction sorbents often result in poor analyte recoveries,

nsufficient cleanup, or irreproducibility from extraction to extrac-ion. Polymeric sorbents are the latest breakthrough in SPE, sincehey enable higher recoveries, higher reproducibility, and loweronsumption of plant materials for the HPLC analysis than classicalorbents. Polymeric sorbents also have the advantage of remain-ng “conditioned” even if the sorbents accidentally run dry duringhe extraction. Divinylbenzene based polymeric sorbents exhibitxcellent stability over the whole pH range unlike classical mod-fied silica gel sorbents C18 and C8 [164]. Excellent results werebtained using polymeric sorbents for concentration and clean-upf isoflavones from red clover [164,165] and soy extracts [126]. Foroy isoflavones, divinylbenzene based polymeric cartridges showedetter retention and much higher breakthrough volume duringample load and washing steps than classical C18 sorbents fromifferent manufactures.

Besides of the use of new polymeric sorbents, the recent trendor the use of SPE is automation and coupling on-line with the anal-sis method. Compared with manual methods, automated SPE isess labour intensive, requires less sample handling providing bet-er recovery, is more reproducible, is performed in a closed systemless chance of sample oxidation or solvent evaporation) and can beerformed relatively fast. For instance, Rostagno et al. [126] devel-ped an automated SPE method for soy isoflavones achieving veryigh recoveries (99.37%) and reproducibility (>98%) with a concen-ration factor of approximately 6:1 in less than 10 min. Anotheruture prospect for the use of SPE is to be coupled on-line with thextraction (such as PLE and SFE) and analysis methods reducingo a minimum post-treatment of extracts in order reduce sampleandling allowing more precise and reliable data to be obtained.

. Separation approaches/techniques

Many different analytical methods can be used for the analysisf isoflavones from soybeans and soy foods. These analytical meth-ds include gas chromatography, liquid chromatography (both withnd without mass detectors) capillary electromigration techniquesCE), and immunoassay. In the last few years several reviews abouthe analysis of isoflavone extracts using these techniques have beenublished [166–171].

Chromatography and CE are, without doubt, the most relevantechniques applied in this field. The use of CE for the analysis ofoy isoflavone samples is very attractive due to the high resolution,fficiency and analysis speed with minimum reagent and sampleonsumption. There are a variety of versatile CE separation prin-iples which are feasible of adapting to solve different analyticalroblems. The possibility of coupling CE to different types of detec-ors, especially to sensitive electrochemical detectors, is one of the

ain advantages of these techniques and point to a powerful toolor the characterization of isoflavones in soy derived samples.

Although the use of CE in the identification process is a poten-ially appropriate means of rapid screening it has been applied onlyn a few occasions for the analysis of isoflavones from soy sam-les, and in most cases only some chemicals forms were identified168,171–174].

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togr. A 1216 (2009) 2–29

However, CE is characterized by poor quantitative reproducibil-ty, mainly caused by inconsistent flow rate and injection volumer amount. Although significant advances in this aspect has beenade, the reproducibility issues of CE, especially when applied to

eal samples, still needs to be solved before it become a real alter-ative to more consolidated techniques such as chromatography171,175].

In this context, of all available analysis techniques, HPLC ishe method of choice since it requires simple pre-analysis samplereparation, allows measurement of all isoflavone chemical forms,

s highly efficient and reproducible, is widely available and has beenxtensively studied. HPLC separation of isoflavones is generally car-ied out on reversed-phase columns with using MeOH or MeCNnd water containing a small amount of acid (formic, acetic, phos-horic or trifluoroacidic acids) as mobile phase. Since isoflavonesxhibit a weak acidic nature the use of acids can make the analyteso be easily dissociated in a solvent system enhancing chromato-raphic separation, resolution and improve peak shape [169]. Mostften used detectors coupled to HPLC are UV and UV-diode arrayetection (DAD) monitoring in the range of 230–280 nm, since all

soflavones exhibit an intense absorption in this UV region of thepectrum. Gradient elution is usually necessary in order to sepa-ate all main isoflavones since they are very chemically close. Asreviously mentioned some isoflavones are particularly difficulto separate from each other (i.e. MGi, AGly and De) [81] and iso-ratic elution has proven to be insufficient. Isocratic elution may beccomplished if a hydrolysis step is used before analysis with themplicit handicap of quantifying only the aglycone forms [51].

Conventional microparticulate 5 �m RP-C18 columns are theost used stationary phase and analysis time needed to separate

ll main soy isoflavones usually reach 60 min. Similarly to sam-le preparation, the current trend for the analysis of soy isoflavonextracts is toward fast, high sensitive and high-resolution separa-ion of all main chemical forms of these compounds in soybeansnd soy foods.

One alternative to achieve faster and more sensitive analysis iso reduce the particle size of the stationary phase. The use of small-article columns (less than 2 �m particle size) can shorten analysisimes, while maintaining – or even increasing – high separation effi-iencies, since it is very well known from Van Deemter equationshat the efficiency of chromatographic processes is proportional toarticle size decrease and to the higher allowed linear velocities.he negative aspect of small particle packed columns used in HPLCs the higher column back-pressure generated [176,177]. Hence, toake full advantage of sub-2 �m particles stationary phases highressure liquid chromatography systems that operate at high pres-ures (>400 bar) are required. Not only is the system capacity ofperating at high pressures important but also the ability to accu-ately and reproducibility integrate an analyte peak and detectorampling rate must be high enough to capture enough data pointscross the peak. Some applications of this innovative technology forsoflavones from different matrixes can be found in the literature178–181].

Churchwell et al. [178] compared UPLC–MS conventionalPLC–MS for the determination of isoflavones in waste water and

ound that in general, UPLC–MS produced significant improve-ents in method sensitivity, speed, and resolution when compared

o conventional HPLC–MS. Improvements in chromatographic res-lution with UPLC were apparent from generally narrower peak androm a separation of diastereomers not possible using HPLC.

As an example of the enormous potential of these new advancesn chromatography, Klejdus et al. [179], developed an analysis

ethod for some selected isoflavones (Di, Gly, Gi, De, Gle, Ge,nonin, sissotrin, formononetin and biochanin) which takes less

han 1 min. The method was successfully applied to soy bits and red

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lover extracts with excellent results. Moreover, Klejdus et al. [180],ccomplished simultaneous separation of not only isoflavones butlso together with several phenolic acids in less than 2 min. Fur-her investigations however, are still needed to evaluate the usef small particle columns for the separation of malonyl and acetylsoflavones that are the most troublesome compounds to separaten soy extracts.

Another alternative to perform high-speed separations usingiquid chromatography is the use of monolithic columns. As com-ared to particle bed columns, monolithic columns represent aingle piece made of porous cross-linked polymer or porous silica.onoliths are made in different formats as porous rods, generated

n thin capillaries or made as thin membranes or disks. The majoroals of applying monolithic columns in HPLC were to achieve lowolumn backpressure and fast mass transfer kinetics [182,183].

Major chromatographic features of monolithic silica columnsrise from the large through-pore size/skeleton size ratios and highorosities, resulting in high permeability and large number of the-retical plates per unit pressure drop. High permeability and smalliffusion path length provided by the presence of large through-ores and relatively small-sized skeletons resulted in the lowerlate height and the lower pressure drop with monolithic silicaolumns compared with a particle-packed column. With lower col-mn backpressure it is possible to increase solvent flow rate making

aster separations possible with current instrumentation [182].Monolithic columns have been used in some occasions for

he analysis of isoflavones in soy extracts [81,110,126,151,184,185].pers et al. [184], achieved separation of most soy isoflavones

except malonyl glucosides) from soy extracts in less than 18 min.urther, Kim et al. [185] recently proposed another method usingonolithic columns for the analysis of four isoflavones (Gi, Ge, Di

nd De) from soybeans and soybean pastes in 7 min.To date, the fastest separation of all main isoflavones in soy

xtracts was obtained by Rostagno et al. [81]. After optimization ofnalysis conditions using monolithic columns all isoflavones wereesolved in less than 10 min using acidified MeOH–water at a flowate of 4 mL/min. Such high solvent flow rate illustrates the lowressure obtained with this type of column.

However, the use of MeOH in the mobile phase to perform fasteparation has some limitations when compared to MeCN, since itas a higher viscosity resulting in higher pressure, which can reduceaximum solvent flow rate allowed within the chromatographic

ystem maximum pressure and increase separation time requiredor all isoflavone chemical forms. Therefore, it is feasible to assumehan even shorter analysis run times than 10 min can be achievedither with monolithic columns with MeCN or with small particleolumns using UPLC systems and that more research is needed inhis direction.

To perform fast HPLC analysis, aside the chromatographic sys-em, the stationary or mobile phase, the simplest approach consistsn operating columns at higher temperatures. Mobile phase vis-osities decrease rapidly with increasing temperature; the columnfficiency is barely changed but the optimum velocity increasesarkedly, allowing the same resolution to be obtained much faster

ut with nearly the same inlet pressure [186]. In this case, some ofhe new small particle columns have a clear advantage over con-entional 5 �m C18 and monolithic columns since they can operatet much higher temperatures, reaching 90 ◦C depending of pH con-itions.

. Conclusions

In the last two decades considerable efforts were directed touantify isoflavones in soybeans and derived foods generating sev-ral sample preparation and analysis methods which resulted in

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togr. A 1216 (2009) 2–29 27

uge amounts of scattered information. Although a critical view ofhese methods is given in this review, is important to point out thathey contributed to better understanding of the complex task thats isoflavone determination and that the necessary steps are beingiven in the direction of achieving reliable and precise informationbout the distribution of these compounds in foods.

Several aspects of sample conservation and sample prepara-ion for the determination of soy isoflavones should be carefullybserved since they are an important source of misinforma-ion. Sample conservation is particularly important since somesoflavones are relatively unstable and adequate storage conditionsre necessary to preserve the original profile of these in soybeansnd soy foods.

Recent trends in sample preparation include automation, high-hroughput performance, reduction in solvent volume and time,n-line coupling with analytical instruments and more impor-antly, reduction of sample manipulation. Although significantdvances on the extraction of isoflavones using modern extrac-ion techniques have been achieved, the full potential of the newechnology available needs to be further explored. There is increas-ng evidence that a combination of extraction/clean-up techniquesuch UAE, PLE and SPE is the most promising application of the newvailable technology for the development of extraction methods forhe determination of isoflavones. Using such combinations not onlyigh-throughput performance and on-line coupling with the analy-is instrument is possible, but also the reduction of post-extractionteps necessary before analysis. Moreover, the availability of newPE sorbents may be easily used in the future to improve the per-ormance of developed combined methods using actual availableechnologies. Another advantage of the use of PLE is the possibilityf re-extraction of the same sample without manipulation whicheduces analytical errors. This characteristic is very interestingince it may be recommendable to perform sequential extractionsup to 5 extractions of the same sample) in order to ensure thatuantitative extractions are achieved. For the extraction procedure,he natural tendency is to use less toxic, expensive and environmen-al friendly solvents, such as ethanol, under optimized conditionshat maximize extraction efficiency achieving fast and quantitativeecoveries.

Another technical tendency in sample preparation is to mini-ize pre and post-extraction steps in order to take full advantage

f fast extraction and analysis procedures and to reduce analyti-al errors due to sample manipulation. This trend coupled to thencreasing availability of commercial standards and higher sen-itivity and resolution of new chromatography technology pointsoward the use of non-hydrolytic methods for the quantification ofoy isoflavones. Furthermore, gathering full information about theistribution and concentration of all main soy isoflavone chemicalorms present in the samples may be critical to understand the rolef these compounds in preventing diseases.

Similarly, for the determination of isoflavones the current trends toward fast, high sensitive and high-resolution separation of all

ain chemical forms of these compounds in soybeans and soyoods. The development of new column packing technology and

aterials as well as of chromatographic systems that can oper-te at high pressures allows analysis time to be drastically reducedrom the usual 60 min to a few minutes with outstanding perfor-

ances showing that further advances can be made in analyticalethodology currently used.Altogether, the optimization of sampling, sample preservation

nd sample preparation parameters are critical for accurate esti-ation of isoflavones present in soybeans and soy foods. Accurate

stimation of isoflavones in soybeans and soy foods, as well as inther samples, will enable researchers to correctly evaluate thenfluence of such phytochemicals on health and provide precise

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ietary and safety guidelines on consumption of these compounds.n addition, accurate quantification of isoflavones in soybeans andoy foods will allow manufacturers, consumers, and marketing pro-essionals to differentiate quality value-added products from theonventional ones. Most importantly, the scientific community,ncluding journal editors and referees, must recognize the necessityf validating sampling, preservation and extraction as critical stepsn describing the content of isoflavones in foods, in order to advancen the field of functional foods for both research and industry.

cknowledgment

The authors acknowledge funding from the Instituto Nacionale Investigación y Tecnología Agraria y Alimentaria (INIA) projectT07-003.

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