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Article Simultaneous Determination of , and in by Capillary Zone Electrophoresis

Feng Luan *, Li Li Tang, Xuan Xuan Chen and Hui Tao Liu College of Chemistry and Chemical Engineering, Yantai University, Yantai 264005, China; [email protected] (L.L.T.); [email protected] (X.X.C.); [email protected] (H.T.L.) * Correspondence: fl[email protected]; Tel.: +86-535-6902063

Academic Editor: Doo Soo Chung Received: 29 October 2016; Accepted: 20 December 2016; Published: 1 January 2017 Abstract: Coffee is a favorite and beverage in Western countries that is consumed daily. In the present study, capillary zone electrophoresis (CE) was applied for the separation and quantification of three isoflavones including daidzein, genistein and formononetin in coffee. Extraction of isoflavones from the coffee sample was carried out by extraction and purification process using ether after the acid hydrolysis with the butylated hydroxy- (BHT). The experimental conditions of the CE separation method were: 20 mmol/L Na2HPO4 buffer solution, 25 kV applied voltage, 3 s hydrodynamic injection at 30 mbar, and UV detection at 254 nm. The results show that the three compounds can be tested within 10 min with a linearity of 0.5–50 µg/mL for all three compounds. The limits of detection were 0.0642, 0.134, and 0.0825 µg/mL for daidzein, formononetin and genistein, respectively. The corresponding average recovery was 99.39% (Relative Standard Detection (RSD) = 1.76%), 98.71% (RSD = 2.11%) and 97.37% (RSD = 3.74%).

Keywords: capillary zone electrophoresis (CE); daidzein; genistein; formononetin; acid hydrolysis

1. Introduction (PEs), also called “dietary ”, are a group of natural, non-steroidal, -like compounds found in a wide variety of consumed by [1]. Currently, four different groups of phenolics are considered PEs, including isoflavones and , coumestanes, flavonoids and stilbenes. Other less-investigated compounds include prenylflavonoids, flavones, flavans and esters [2]. These compounds are similar to 17β- in structure and function [3]. They have a dual-directional regulation function in the body, meaning both anti-estrogen activity and estrogenic activity [4]. Such compounds have good curative effects for the treatment of , , Alzheimer’s disease, neurodegenerative disorders, and , etc. [5–8]. They can also be used for the treatment of the symptoms of [9]. PEs are common in nature and have many pharmacological activities [9,10]. It has been reported that the daily take of PEs for menopausal women in the Caucasus region of the United States is no more than one milligram [11]. There are some foods that contain PEs including: and soy products, , linseeds, sesame seeds, wheat berries, fenugreek, oats, barley, , lentils, yams, rice, alfalfa, mung beans, apples, carrots, pomegranates [12], wheat germ, rice bran, lupin, , coffee, licorice root, mint, , hops [13], bourbon, beer [14], fennel and anise [15]. As we know, the content of PEs is different between various foods. In addition, it may also occur in the same kinds of foods, such as soy beverages and , since they are made of different kinds of raw materials or using different processing methods [16]. It has been proved that -rich diets include soybeans and products, followed by , nuts, oilseeds, fruits and vegetables [17]. As is well known, there is a phenomenon

Separations 2017, 4, 1; doi:10.3390/separations4010001 www.mdpi.com/journal/separations Separations 2017, 4, 1 2 of 8

It has been proved that phytoestrogen-rich diets include soybeans and soybean products, followedSeparations 2017 by , 4 legumes,, 1 nuts, oilseeds, fruits and vegetables [17]. As is well known, there 2 is of a 8 phenomenon where people consume less soy and its derivatives in Western countries than in whereEastern people ones [ consume18]. However, less soy coffee and is its a derivatives favorite beverage in Western in Western countries countries than in that Eastern is consumed ones [18]. daily;However, for example, coffee is a in favorite the United beverage States, in Western29 September countries is celebrated that is consumed as “Nat daily;ional forCoffee example, Day” in [19 the]. Thus,United it States,is necessary 29 September to study is the celebrated content of as “National Coffee in coffee. Day” [19]. Thus, it is necessary to study the contentSome researchers of isoflavones have in coffee.been studied the phytoestrogens in plants [20]. The methods used for the determinationSome researchers of phytoestrogens have been studied in the plants phytoestrogens are GC-MS in[21,22 plants], HPLC [20]. The [17,23 methods–31], andused LC for-MS the [32,33determination]. It should of be phytoestrogens noted that HPLC in plants separation are GC-MS is generally [21,22 carried], HPLC out [17 on,23 reversed–31], and-phaseLC-MS columns [32,33]. usingIt should the bemethanol noted that of acetonitrile HPLC separation and water is generally containing carried small out amounts on reversed-phase of acid as a columns modifier using [29]. theAs farmethanol as we of know, acetonitrile few studies and water have containing been reported small amounts using capillary of acid as zone a modifier electrophoresis [29]. As far (CE) as we in know, the analysisfew studies of haveisoflavones. been reported Aramendia using et capillary al. [34] zone separated electrophoresis and identified (CE) in several the analysis isoflavones of isoflavones. using AramendiaCE-ESI/MS etin al. negative [34] separated ion mode and identifiedfrom standard several samples. isoflavones García using-Villalba CE-ESI/MS et al. in [35 negative] used ioncapillary mode electrophoresisfrom standard samples.–time-of-flight García-Villalba-mass spectrometry et al. [35] used to compare capillary the electrophoresis–time-of-flight-mass metabolic profiles of conventional andspectrometry genetically to compare modified the soybeans, metabolic including profiles of isoflavones, conventional amino and genetically acids, carboxylic modified acids soybeans, and peptides.including isoflavones,In the above amino references, acids, carboxylic separation acids was and carried peptides. out in In positive the above mode references, at pH separation 9–9.5 and wasdetection carried was out achieved in positive in modenegative at pHionization 9–9.5 and mode, detection but quantification was achieved inwas negative not reported. ionization Also, mode, the processbut quantification is somewhat was time not- reported.consuming Also,, though the process it is useful is somewhat and effective. time-consuming, though it is useful and effective.From the works reported so far, one can see that the quantitative determination of isoflavones in plantFroms is themostly works done reported by HPLC so far, combined one can with see thatacid the hydrolysis quantitative or determination extraction. of In isoflavones addition, thein plants separation is mostly system done is acid by HPLCic. The combined HPLC method with aciduses hydrolysis a large amount or alcohol of organic extraction. solvent In in addition, mobile phasethe separation and requires system a long is acidic. separation The HPLC time. method The acid uses hydrolysis a large amount and of organic extraction solvent are in simple mobile; however,phase and the requires process a longcannot separation transform time. The acid into hydrolysis aglycone. and Also, ethanol the acid extraction hydrolysis are process simple; mayhowever, lead the to process the samples cannot contain transforming glycosidesa large number into aglycone. of complex Also,es theand acid it ishydrolysis difficult process to separate may substanceslead to the samplesafter they containing are treated a by large acid number hydrolysis. of complexes and it is difficult to separate substances afterThe they aim are treated of this by work acid was hydrolysis. to develop a simple, efficient and sensitive CE method, as an alternativeThe aim to ofother this workmethods was, tofor develop the quantitative a simple, efficientanalysis andof three sensitive isoflavones, CE method, including as an alternative daidzein, genisteinto other methods, and formononetin for the quantitative (their chemical analysis structures of three isoflavones,are shown in including Figure 1 daidzein,) in coffee. genistein For this, andwe improvedformononetin the (their extraction chemical method structures of isoflavones are shown from in Figure the coffee1) in coffee. sample For with this, an we extracti improvedon and the purifextractionication method process of using isoflavones ether frominstead the of coffee ethanol sample after with the an process extraction of acid and hydrolysis purification with process the antioxidantusing ether butylatedinstead of hydroxy ethanol- aftertoluene the (BHT). process The of acid proposed hydrolysis method with was the successfully antioxidant applied butylated to thehydroxy-toluene determination (BHT).of three The isoflavones proposed in methodcommercially was successfully available coffee applied samples. to the To determination our knowledge, of therethree isoflavones are no methods in commercially that describe available the quantification coffee samples. of isoflavones To our knowledge, in coffee there samples are no using methods the singlethat describe CE. Also, the the quantification sample preparation of isoflavones of this in method coffee samples is simple using, the theanalytical single CE. time Also, is shorter, the sample and thepreparation comparative of this method cost is isrelatively simple, the lower. analytical It can time be is shorter, used as and an the alternative comparative method cost is relativelyfor the determinationlower. It can be of used isoflavones as an alternative in coffee.method for the determination of isoflavones in coffee.

OCH3 OH OH O OH O O

HO HO HO O O O (a) Formononetin (b) Genistein (c) Daidzein

Figure 1. TheThe chemical chemical structures of ( a) formononetin, ( b) g genistein,enistein, and ( c) daidzein.

2. Materials and Methods 2. Materials and Methods

2.1. Chemicals and Reagents IsoflavoneIsoflavone standards standards daidzein (daidzein (≥98%), (≥98%), genistein genistein (≥98%) (≥98%) and formononetin and formononetin (≥98%) were (≥98%) purchased were purchasedfrom Shanghai from Juyuan Shanghai Biological Juyuan Technology Biological Technology Co., Ltd. (Shanghai, Co., Ltd. (Shanghai, China). Disodium China). Disodium hydrogen hydrogen phosphate (Na2HPO4) was obtained from Sinopharm Chemical Reagent Co., Ltd. phosphate (Na2HPO4) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Methanol, acetonitrile were obtained from Kemel Chemical Reagent Co., Ltd. (Tianjin, China). Separations 2017, 4, 1 3 of 8

Butylated hydroxy-toluene (BHT) was used as antioxidant and was obtained from Shanghai Feige Chemical Co. Ltd. (Shanghai, China). The analytical grade regents were used to prepare the samples and the buffer solutions. The water used in all studies was ultrapure water (18.25 MΩ·cm) obtained from an ultrapure purification system (Yongjieda, Hangzhou, China). The nitrogen used for evaporation of solvents was of 99.9% purity and supplied by Feiyuan Comprehensive Service Technology (Yantai, China).

2.2. Standard and Sample Preparation Each type of isoflavone stock solution (1 g/L) was prepared in methanol and stored at −18 ◦C in volumetric flask. A series of calibration solutions (0.5, 1.0, 5.0, 10.0 and 50.0 µg/mL) was prepared by diluting the stock standard solution with methanol and stored at 4 ◦C. Ground coffee, purchased from the local supermarket of United States of America, was passed through a 0.45 mm sieve. A 0.2 g aliquot of ground coffee was spiked with the antioxidant (1%, 50 µL), ethanol (2 mL) and hydrochloric acid 3.4 mol/L HCL (2 mL). The acid hydrolysis was performed at 75 ◦C for 150 min, under reflux. This process was performed according to Alves et al. [23] with minor modification. After the hydrolysis, 1 mL of the hydrolysate was neutralized with 120 µL of sodium hydroxide (10 mol/L), vortexed and ultra-centrifuged (5000 r/min, 5 min). Then 2 mL supernatant was collected, extracted and purified by ether [36]. Then the superstratum ether was dried by nitrogen. The residue was dissolved in methanol, and volume was completed till 2 mL. And then it was filtered through a 0.45 µm Millipore filters before analysis and stored under refrigeration (4 ◦C) until use.

2.3. CE Apparatus In present study, the experiments were performed on a LUMEX CAPEL 105M Capillary Electrophoresis System (LUMEX Ltd., St. Peterburg, Russia). A 60 cm (50.5 cm effective length), 75 µm i.d. fused silica capillary (Yongnian Ruifeng Chromatographic Devices Limited Company, Baoding, China) was used. A PH5500 dual-channel pH/ion meter (Crison, Singapore) was used for regulating the pH of the buffers.

2.4. Electrophoretic Conditions Before using, each new capillary column was rinsed sequentially with ultrapure water for 10 min, 0.5 mol/L NaOH for 40 min, and then ultrapure water for 10 min. At the beginning of each day the capillary was rinsed with ultrapure water for 2 min, 0.2 mol/L NaOH for 10 min, ultrapure water for 5 min and running buffer for 10min. Between different buffers, the capillary was conditioned with ultrapure water for 2 min, 0.2 mol/L NaOH for 10 min, ultrapure water for 5 min, and buffer for 5 min. The capillary was conditioned only with running buffer for 3 min between consecutive injections. All buffers and solutions were filtered through a 0.45 µm Millipore filter before injection. Pressure injection was set at 30 mbar for 3 s. The conditions of electrophoresis was kept the temperature at 25 ◦C, applied voltage at 25 kV, and the UV detection at 254 nm.

3. Results and Discussion

3.1. Optimization of the CE Method It was necessary to determine the parameters of CE before analyzing the daidzein, genistein and formononetin in the coffee sample. The UV detection wavelength of isoflavones is generally 250–265 nm. In order to determine the optimal wavelength for simultaneous determination of the three compounds, the UV absorption spectrum of the isoflavone solution from 230 to 350 nm was scanned (see Figure2). As can be seen from the figure, the best absorption wavelengths of the three compounds were 250–260 nm, 250–260 nm, and 260–270 nm, respectively. However, all three isoflavones have a strong absorption at 254 nm, so the detection wavelength was set to 254 nm. Separations 2017, 4, 1 4 of 8 Separations 2017, 4, 1 4 of 8 Separations0.55 2017, 4, 1 4 of 8 0.50 b c 0.55 a:Daidzein 0.45 c 0.50 b b:Formononetin a:Daidzein 0.40 0.45 a b:Formononetinc:Genistein a 0.35 0.40 c:Genistein 0.30 0.35 0.30 AU 0.25 AU 0.25 0.20 0.20 0.15 0.15 0.10 0.10 0.05 0.05 0.00 0.00 -0.05 -0.05 230 240 250 260 270 280 290 300 310 320 330 340 350 230 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength(nm) Wavelength(nm) Figure 2. UV spectra of formononetin, daidzein and genistein. FigureFigure 2.2. UV spectra of formononetin,formononetin, daidzeindaidzein andand genistein.genistein. Five different concentrations of Na2HPO4 solution at 10, 20, 30, 40 and 50 mmol/L were FiveFiveinvestigated different different in concentrations concentrationsthe present study. ofof With Na Na 2the2HPOHPO buffer44 solution solution atconcentration at 10, 10, 20, 20, 30, 30, increased, 40 40 and and the 50 50 degree mmol/L mmol of/ L werewere investigatedinvestigatedseparation inin thetheimproved presentpresen, andt study.study. at the WithWith same thethe time bufferbuffer the analysis solutionsolution time concentrationconcentration increased as wellincreased,increased,. However, thethe the degreedegree ofof separationseparationanalysis improved,improved time of 30, and mmol/L and at atthe was the same a samelittle time shorter time the analysis than the analysisthat timeof 20 increasedmmol/L. time increased So, as a well.buffer as However,concen well.tration However, the of analysis the 30 mmol/L buffer concentration was selected. time of 30 mmol/L was a little shorter than that of 20 mmol/L. So, a buffer concentration of 30 mmol/L analysis timeThe of buffer 30 mmol/L pH may was have a somelittle shorter effect on than the migration.that of 20 Usingmmol/L. the aboveSo, a buffer optimiz concened buffertration of buffer30 mmol/L concentration buffer concentration (30 was mmol/L selected. Na was2HPO selected.4), the pH was regulated at 7.0, 8.0, 9.0, 10.0, 11.0. As the pH TheTheincreased, buffer buffer the pH pH ana may maylytical have have time some someincreased effect effect; however on on the the, the migration. migration. separation was Using still the not ideal. above above It optimizedwas optimiz almosted bufferbuffer concentrationconcentrationthe same (30 (30as that mmol/L mmol/L of the basic Na22HPO HPO30 mmol/L44),), the the Na pH pH2HPO was was4. So regulated regulated the basic atsystem at 7.0, without 8.0, 9.0, regulating 10.0, 10.0, 11.0. 11.0. the AspH thethe pH pH increased,increased,value thethe was analyticalana usedlytical in the time timeanalysis. increased; increased however,; however the, the separation separation was was still still not not ideal. ideal. It was It was almost almost the In the following, three organic modifiers (methanol, acetonitrile and acetone) were added to 30 samethe same as that as ofthat the of basic the basic 30 mmol/L 30 mmol/L Na2HPO Na24HPO. So4 the. So basic the basic system system without without regulating regulating the pH the value pH mmol/L Na2HPO4 at pH = 9.9 to investigate the influence of the separations, respectively. From the wasvalue used was in used the analysis.in the analysis. results, we can see that their presence leads to no obvious improvement in the peak shape. InInTherefore, the following, none of three threethe organic organic organic modifier modifiers modifierss was added (me (methanol,thanol, to the separation acetonitrile acetonitrile system. and and acetone) acetone) were were added added to to30 30mmol/L mmol/L NaThe Na2HPO 2effectsHPO4 at 4of pHat an pH =injection 9.9 = 9.9to toinvestigatetime investigate of 1, 3, 5,the 8 the and influence influence 10 s at 30of ofmbar the the separation were separations, also investigateds, respectively respectively. in order. From thethe results,results,to we we obtain can can seea better see that that sensitivitytheir their presence andpresence peak leads shapeleads to no; finally obvious to no, 3 obviouss improvement proved improvement to be ideal in the for peak the in subsequenshape.the peak Therefore,t shape. noneTherefore, ofanalysis. the none organic Similarly, of the modifiers organic effects was ofmodifier temperature addeds was to the (in added theseparation range to the of system.separation 15–25 °C ) and system. applied voltage (in the TheTherange effectseffects of 15 of–of25 anan kV)injection injection on the separation time time of of 1, efficiencies1, 3, 3, 5, 5, 8 8 and and were 10 10 salso ats at 30verified. 30 mbar mbar Finally, were were also a alsotemperature investigated investigated of 25 in °C orderin order to and an applied voltage of 25 kV were selected. obtainto obtain a better a better sensitivity sensitivity and peakand peak shape; shape finally,; finally 3 s proved, 3 s proved to be ideal to be for ideal the subsequent for the subsequen analysis.t From the above results, the optimal CE separation◦ conditions were selected: 30 mM Na2HPO4, Similarly,analysis.3 s effectsSimilarly,pressure of injection temperature effects at of30 temperaturembar, (in the 25 range kV applied of(in 15–25 the voltage rangeC) andat of25 applied 15°C– 25and °C voltagedetection) and (in appliedat the254 rangenm. voltage Under of 15–25 (in kV) the ◦ onrange the separationofthese 15 –conditions,25 kV) efficiencies on the the electropherogram separation were also efficiencies verified. of daidzein, Finally, were genistein aalso temperature verified.and formonon ofFinally, 25etinC wasa and temperature obtained an applied and of voltage 25 °C ofand 25 an kVis applied shown wereselected. in voltage Figure 3. of 25 kV were selected. FromFrom thethe aboveabove results,results, thethe optimaloptimal CECE separationseparation conditionsconditions werewere selected:selected: 3030 mMmM NaNa2HPOHPO4, 6 2 4 33 ss pressurepressure injection injection at at 30 30 mbar, mbar, 25 25 kV kV applied applied voltage voltage at 25 at◦ C25 and °C detectionand detection at 254 at nm. 254 Under nm. Under these conditions,these conditions, the electropherogram the electropherogram4 D-Daidzein of daidzein, of daidzein, genistein genistein and formononetin and formonon wasetin obtained was andobtained is shown and inis shown Figure3 in. Figure 3. G-Genistein 2 F-Formononetin

mAu 6 0 D F G 4 D-Daidzein -2 G-Genistein 2 5.0F-Formononetin5.5 6.0 6.5 7.0 7.5 8.0 (min) mAu Time Figure 3. Electropherogram of the standard mixture of formononetin, daidzein and genistein. 0 F D G -2

5.0 5.5 6.0 6.5 7.0 7.5 8.0 Time (min) Figure 3. Electropherogram of the standard mixturemixture ofof formononetin,formononetin, daidzeindaidzein andand genistein.genistein.

Separations 2017, 4, 1 5 of 8

3.2. Validation of the CE Method

3.2.1. Linearity, Limit of Detection and Limit of Quantification The linear ranges were tested at 0.5–50.0 µg/mL for the three isoflavones at five concentration levels. Calibration curves were obtained by plotting the peak area of each standard as a function of the standard concentration (C). The correlations were: y = 4.0245C + 2.3062 (R2 = 0.9995) for formononetin; y = 8.4133C − 2.8224 (R2 = 0.9998) for daidzein; y = 6.5477C − 1.3905 (R2 = 0.9994) for genistein. The limit of detection (LOD) and limit of quantification (LOQ) were determined as 3 σ/s and 10 σ/s, respectively, where σ is the standard deviation of the blank responses and s is the slope of the calibration curve. The LODs of formononetin, daidzein and genistein were 0.134, 0.0642 and 0.0825 µg/mL, respectively. The LOQs were 0.446 µg/mL for formononetin, 0.214 µg/mL for daidzein, and 0.275 µg/mL for genistein.

3.2.2. Study of Precision and Accuracy The precision of the proposed method was validated by the intra-day and inter-day values of the migration time and peak area under the optimal conditions. The intra-day repeatability was determined by replicating injections of a 10 µg/mL solution of isoflavones six times in one day, while the inter-day repeatability was determined by performing injections for six consecutive days. The results are listed in Table1. As can be seen in the table, the relative standard deviation (RSD) was less than 1.6% for the migration time and less than 5.4% for the peak area in all cases.

Table 1. Intra-day and inter-day precision of migration time and peak area for isoflavones.

Intra-Day (%) Inter-Day (%) Compound Migration Time Peak Area Migration Time Peak Area Formononetin 0.34 4.60 0.55 5.31 Daidzein 0.62 3.45 1.23 4.51 Genistein 1.03 4.29 1.52 5.01

The method’s accuracy was evaluated by the standard addition procedure. Firstly, blank samples were analyzed in order to determine the actual content of the three isoflavones in the sample. Then, three different amounts (0.5 µg/mL, 1.0 µg/mL and 2.0 µg/mL) of isoflavones were added into three coffee samples before the extraction. The results are presented in Table2. From the table, we can see that satisfactory average recoveries (97.37%–99.39%) were obtained with an RSD lower than 3.74%. These data show that the pretreatment method has good extraction efficiency.

Table 2. Recoveries for the determination of the three isoflavones in coffee.

Concentration Added Recovery RSD Compound (µg/mL) (%, n = 3) (%) 0.5 105.20 Formononetin 1.0 95.53 1.76 2.0 97.44 0.5 92.31 Daidzein 1.0 103.34 2.11 2.0 100.48 0.5 103.58 Genistein 1.0 90.87 3.74 2.0 97.66 Separations 2017, 4, 1 6 of 8

Separations 2017, 4, 1 6 of 8 3.3. Optimization of the Extraction Conditions and Sample Analysis Pretreatment process optimization has to be developed since the isoflavones occur mainly as glycosidesPretreatment present process in the coffee optimization [37,38]. For has the to bepurpose developed of detecting since the the isoflavones total amount occur of aglycones, mainly as glycosidesthe acid procedure present in the is usually coffee [37 performed.,38]. For the According purpose of to detecting Alves theet al. total [23] amount, acid of hydrolysis aglycones, was the acidperformed procedure first is in usually the present performed. study. According However, to it Alves cannot et give al. [23 an], acidideal hydrolysis detection wasusing performed the same firstprocedure in the present as in the study. refer However,ence. So it we cannot extracted give an and ideal purified detection the using sample the with same ether procedure [36] after as in the the reference.process of So we acid extracted hydrolysis and purified with the the sample antioxidant with ether butylated [36] after hydroxy the process-toluene of acid (BHT). hydrolysis The withelectropherograms the antioxidant of butylated the coffee hydroxy-toluene samples are shown (BHT). in Figure The electropherograms 4. From the figure, of one the can coffee see samples that the aredaidzein shown can in Figurebe detected4. From and the at figure, a value one of can 0.012 see thatmg/g. the C daidzeinompared can with be detectedthe results and of at the a valuework of of 0.012Alves mg/g. et al. [23], Compared we can with see thethat results the amount of the workof daidzein of Alves found et al. in [23 this], we study can seeis in that accordance the amount with of daidzeinthe value found reported in this by studythe HPLC is in method. accordance The with work the of value Kuhnle reported et al. [31] by the reported HPLC total method. isoflavones The work of of1 mg Kuhnle per 100 et al. g [of31 instant] reported coffee total which isoflavones is also ofsimilar 1 mg to per our 100 results. g of instant The other coffee two which isoflavones is also similar could tonot our be results.detected The in otherour study two.isoflavones The reason couldmay be not that be dif detectedferent inkinds our of study. coffee The may reason contain may different be that differentquantities kinds or kinds of coffee of isoflavones may contain [23 different], or the quantities contents orof kinds the two of isoflavones could not be[23 ], detecte or thed contents for this ofparticular the two couldsample not. There be detected are other for thisworks particular [21,39] that sample. have There detect areed other the coffee works beverage [21,39] that (1 haveìg of detectedisoflavones the per coffee cup beverage of coffee (1 beverage ìg of isoflavones (287 mL)) per and cup coffee of coffee brew beverage (10–66 ì (287g of mL))daidzein, and coffee15–29 brewìg of (10–66genistein, ìg of and daidzein, 72–78 ìg 15–29 of formononetin ìg of genistein, for a and 100 72–78 g cup ìg of of coffee formononetin brew) values for, a which 100 g cupindicated of coffee our brew)results values, are acceptable. which indicated our results are acceptable.

6 a D-Daidzine G-Genistein F-Formononetin 4

mAu D

2

5.0 5.5 6.0 6.5 7.0 7.5 8.0

Time (min)

2 b

0 mAu F D G -2

5.0 5.5 6.0 6.5 7.0 7.5 8.0 Time (min)

FigureFigure 4. 4.Electropherograms Electropherograms of (a of) coffee (a) coffee sample; sample (b) coffee; (b) sample coffee spiked sample with spiked 1.0 µg/mL with three 1.0 µg/mLisoflavones. three isoflavones. 4. Conclusions 4. Conclusions This work presents the application of CE for the determination of daidzein, genistein and This work presents the application of CE for the determination of daidzein, genistein and formononetin in coffee. This method has the advantages of high sensitivity, shorter analytical time, formononetin in coffee. This method has the advantages of high sensitivity, shorter analytical time, minor amounts of samples and simple operation. It is concluded that the CE method is a good minor amounts of samples and simple operation. It is concluded that the CE method is a good alternative to other methods. alternative to other methods. Acknowledgments: This work was financially supported by the National Natural Science Foundation of China (No.Acknowledgments: 21675138). This work was financially supported by the National Natural Science Foundation of China Author(No. 21675138). Contributions: Feng Luan and Huitao Liu conceived and designed the experiments; Xuanxuan Chen and LiliAuthor Tang Cont performedributions: the experiments;Feng Luan and Feng Huitao Luan L andiu conceived Xuanxuan and Chen designed wrote the the paper. experiments; Xuanxuan Chen Conflictsand Lili Tang of Interest: performedThe the authors experiments; confirm thatFeng this Luan article and contentXuanxuan has C nohen conflict wrote of the interest. paper.

Conflicts of Interest: The authors confirm that this article content has no conflict of interest. References

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