Rapid magnetic solid-phase extraction for the selective determination of isoflavones in soymilk using baicalin-functionalized magnetic nanoparticles (2025)

Keywords: soymilk, sample preparation, baicilin-functionalized magnetic nanoparticles, magnetic solid phase extraction, HPLC–ESI-MS/MS

Reagents and Chemicals

Soymilk samples were purchased from a local market in Madison, MS, USA and stored at 4 °C till analysis. HPLC grade acetonitrile and formic acid were purchased from Fisher Scientific (Hanover Park, IL). Baicalin was prepared from Scutellaria baicalensis in our laboratory and its structure was elucidated on the basis of MS, ¹H NMR and ¹³C NMR spectral evidences. Six isoflavone standards daidzein (De), glycitein (Gle), genistein (Ge), daidzin (Di), glycitin (Gli), genistin (Gi) and internal standard calycosin (IS) (structures shown in Figure 1) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Milli-Q (Millipore Corp., Bedford, MA) water was used throughout the work. Other chemicals and solvents of analytical grade were purchased from Sigma–Aldrich Chemical (St. Louis, MO, USA).

Preparation of Baicalin-functionalized Magnetic Nanoparticles (BMNPs)

The multistep procedure for the preparation of BMNPs is shown in Fig. 2. First, amino-functionalized magnetic nanoparticles (AMNPs) were prepared by hydrothermal method as follows: 6.5 g of 1,6-hexanediamine, 2.0 g of anhydrous sodium acetate and 1.0 g of FeC₃ 6H₂O were dissolved in 30 mL of ethylene glycol by vigorously stirring at 50 °C to get a transparent solution. Then, the mixed solution was transferred into a teflon lined autoclave to obtain AMNPs for 6 h at 198 °C. The AMNPs were then rinsed with water and ethanol twice, and then dried at 50 °C. During each rinsing step, the AMNPs were separated from the supernatant by applying an external magnet.³⁸

Finally, 150 mg AMNPs and 50 mg bacialin were dispersed in 10 mL 75% dimethyl sulfoxide (DMSO)/phosphate solution (10 mM, pH 5.5) containing 35 mg 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS). After gently shaking for 2 h, washed by 75% DMSO and water twice successively, BMNPs were obtained by magnetic separation. The preparation procedures are illustrated in Figure 2. The loading mass of baicilin in BMPNs was characterized by elemental analyzer.

BMNPs Characterization

Transmission electron microscope (TEM) images were acquired on a Tecnai G² F20 S-TWIN TEM (200 KeV, FEI, OR, USA). Carbon and nitrogen analyses were performed on a Carlo Erba (Italy) model 1106 elemental analyzer.

MSPE Procedure

To a tube containing 100 μL soymilk sample 5 μL of calycosin at 40 mg/L was added as internal standard (IS). While stirring, 100 μL BMNPs solution prepared above was added. The mixture was vigorously shaken for 5 min using a vortex oscillator. The tube was placed on a magnet for 30 sec to let BMNPs settle down. The supernatant was discarded. After 2 times washing with 100 μL water, isoflavones were eluted out from BMNPs with 100 μL warm methanol (about 60 °C). After magnetic separation, the supernatant was carefully saved, and mixed with 100 μL water. Portions (20 μL) were injected into the HPLC–ESI-MS/MS system for analysis without further purification. The MSPE procedure described above is illustrated in Figure 3.

HPLC–ESI-MS/MS Analysis

The system consisted of two pumps (LC-10ADvp, Shimadzu, Toyoto, Japan), an on-line degasser (DGU-12A, Shi-madzu), and triple quadrupole mass spectrometers equipped with an ESI source (TSQ Quantum, Thermo Scientific, San Jose, CA, USA). Both the LC and mass spectrometer were controlled by Xcalibur software (Thermo Finnigan). A reversed-phase column (C₁₈, 150 mm × 2.1 mm, 5 μm) was used for separation. A switching-valve was placed after the column directing the effluent either to waste or to the MS detector. Gradient LC elution was carried out with two mobile phases: (A) 10% acetonitrile/0.1% formic acid and (B) 90% acetonitrile/0.1% formic acid at a flow rate of 0.25 mL/min. The elution was programmed as following: time 0–20.00 min, mobile phase B was linearly increased from 10% to 45%; time 20.10–25.00 min, 100% mobile phase A to equilibrate the column for next run.

The MS detector was operated in the positive mode with the following settings: spray voltage of 3 KV, vaporizer temperature of 300°C, tube lens voltage of 150 V; capillary voltage of 35 V, capillary temperature of 270°C, and sheath and aux gas flow rates of 35 and 10, respectively. The optimized relative collision energies for collision-induced dissociation (CID) were 30 V, using m/z 255→199 for daidzein, m/z 271→153 for genistein, m/z 285→242 for glycitein and calycosin (IS), m/z 417→255 for daidzin, m/z 433→271 for genistin, and m/z 447→285 for glycitin SRM detection, respectively.

Characterization of BMNPs

A facile two-step synthesis of BMNPs was developed in this work. Firstly, AMNPs were prepared by one-pot strategy using FeC₃ 6H₂O and 1,6-hexanediamine as reported previously.³⁸ Then, the acid amide was condensed through the –NH₂ on AMNPs and the –COOH on baicalin using EDC and Sulfo-NHS as dehydrating agents. Sulfo-NHS was used to prepare amine-reactive esters of carboxylate groups for crosslinking, i.e. carboxylates (-COOH) of baicalin reacted to Sulfo-NHS in the presence of a carbodiimide (here is EDC), to form a semi-stable Sulfo-NHS ester, the latter of which then reacted with primary amines (-NH₂) to form amide crosslinks (as shown in Figure 2).

The TEM image of BMNPs with 400 nm in diameter is shown in Figure 4. According to the BMNPs structure (Figure 2), the carbon came from baicalin and AMNP. And the ratio of carbon to nitrogen in AMNPs part was constant (3:1). Thus, the mass percentage of loaded baicalin in BMNPs was calculated as 5.13±0.06% which was determined indirectly by the quantities of carbon and nitrogen using Eq. (1), in which C% and N% are the content of carbon and nitrogen in BMNPs; Ar (C), Ar (N) and Mr (C₂₁H₁₈O₁₁) are the relative atomic mass of carbon and nitrogen, and relative molecular mass of baicalin, respectively.

MSPE Procedure

Baicalin-functionalized magnetic-nanoparticles (BMNPs) may serve as effective magnetic SPE sorbents for selective extraction of isoflavones. The affinity may be attributed to the following factors: the polar functional groups in baicalin molecule, such as –OH and –CHO–, can form hydrogen bonds with isoflavones; the planar structure of baicalin molecular skeleton (C₆-C₃-C₆, a great conjugated system) is prone to absorb similar planar structures through π–π and/or n–π charge transfer effects; and the great conjugated π–π system in baicalin molecule enhances the charge transfer capacity through the increment of the electron density in the ring. Therefore, the interaction of BMNPS with polar aromatic compounds such as isoflavones is particularly strong. The interaction is comparable to SPE cartridges based on divinylbenzene, ³⁹ which are very effective to selectively extract soy isoavones. In the present MSPE procedure, sorbents (BMNPs) were added into the soymilk solution. The targeted analytes (isoflavones) in the soymilk were adsorbed onto the sorbent surface under vigorously shaking. The sorbents with captured analytes were then easily recovered from the suspension using an external magnetic separator. The analytes were consequently eluted from the sorbents and analyzed by HPLC–ESI-MS/MS. Compared to widely used freeze-dried methods, the application of MSPE greatly simplified the sample pretreatment which needed no more than 10 min for sample preparation.

Daidzein, one of the isoflavones in soymilk, was selected as the model compound to evaluate the extraction efficiency. The extraction efficiencies remained almost the same for extraction times of 5, 10, and 20 min (from 88.7±0.2% to 89.5±0.3%); while the eluting efficiencies remained almost the same for elute times of 1, 3, and 5 min. Therefore, 5 min and 1 min were selected for extraction and eluting time, respectively. Isoflavones are soluble in methanol, ethyl acetate, pyridine, slightly soluble in warm water, and practically insoluble in chloroform and hexane. Therefore, water-based soymilk was directly used as the loading solvent. On the other hand, warm methanol at about 60°C was chosen as eluting solvent due to its high miscibility with water as well as high suitability for HPLC analysis. To improve the accuracy and reproducibility of the quantification of isoflavones in soymilk, calycosin (IS), an isomer of one of the isoflavones targeted (i.e. glycitein) was used as internal standard. Calycosin exhibited behaviors similar to the isoflavone analytes in both MSPE and MS detection processes. Calycosin does not occur naturally in soy beans. It is also easily available compared with isotope labeled isoflavones. An equal amount of water was added into the elution solution prior to HPLC–ESI-MS/MS analysis to minimize the solvent effects on the separation.

Analytical Figures of Merit

After the sample preparation, isoflavones in soymilk were quantitatively analyzed using calycosin as internal standard (IS). LC–MS analysis of authentic daidzein, genisetin, glycitein, daidzin, genistin, glycitin and internal standard was performed. The behaviors of chromatographic retention and product ion spectra of these six isoflavones are shown in Figure 5. Using m/z 255→199 for daidzein, m/z 271→153 for genistein, m/z 285→242 for glycitein and calycosin (internal standard), m/z 417→255 for daidzin, m/z 433→271 for genistin, and m/z 447→285 for glycitin SRM MS/MS mode, the quantification was carried out by means of the signal ratio of analyte to internal standard. Five-point calibration curves were prepared with authentic isoflavones solutions at concentrations ranging from 0.3 mg/L to 80 mg/L while keeping internal standard concentration constant at 1 mg/L. Peak areas were used for the calculation. Linear regression analysis of the results yielded the following equations for the six isoflavones:

$$\begin{array}{lll}\text{daidzein}\hfill & \text{Y}=0.8505\text{X}+0.0366\hfill & r^2=0.993\hfill \\ \text{genistein}\hfill & \text{Y}=1.0713\text{X}+0.0323\hfill & r^2=0.995\hfill \\ \text{glycitein}\hfill & \text{Y}=0.6809\text{X}+0.0230\hfill & r^2=0.993\hfill \\ \text{daidzin}\hfill & \text{Y}=5.0499\text{X}+0.0650\hfill & r^2=0.998\hfill \\ \text{genistin}\hfill & \text{Y}=5.1745\text{X}+0.0560\hfill & r^2=0.994\hfill \\ \text{glycitin}\hfill & \text{Y}=4.9001\text{X}+0.0183\hfill & r^2=0.997\hfill \end{array}$$

where Y is the peak area ratio of the analyte to internal standard, X is the concentration of the analyte in mg/L, and r² is the correlation coefficient. Interday (5 days) precisions of the slope and intercept of the calibration curves were found to be in the range between 2.5% and 3.6% (RSD, n = 5). From the calibration curves, the limits of detection were estimated to be in the range from 0.03 mg/L for genistin to 0.05 mg/L for glycitein (signal/noise = 3). These results indicated that the present method was sufficiently sensitive for the analysis of isoflavones in soymilk. Comparing with other sample preparation approaches previously reported for soymilk analysis, the present MSPE procedure was easy to carry out and much faster (less than 10 min). As shown in Table 2, analytical figures of merit are compared for several HPLC-based quantitative methods reported^{31, 32, 40} for analysis of isoflavones in soymilk.

HPLC–ESI-MS/MS determination of isoflavones in soymilk samples

Monitoring the levels of isoflavones in commercial soymilks is of importance. Using the present HPLC–ESI-MS/MS method, six isoflavones (daidzin, genistin, glycitin, daidzein, genistein, and glycitein, respectively) were detected and quantified simultaneously from soymilk samples obtained from local supermarkets. Typical chromatograms from these analyses are shown in Figure 6. As can be seen, peaks corresponding to isoflavones were well identified. Besides, a minor peak with same MS and MS² behavior to genistein was observed at retention time 5.2 min (Figure 6). Because it was well separated from genistein by HPLC, it didn’t interfere with the quantification. All the analyses were conducted three times, and the analytical results are summarized in Table 1. Isoflavone concentrations in the soymilk samples were found at the mg/L level which was consistent with the results reported previously.⁴¹ Recovery of this isoflavones from soymilk matrix by the present HPLC-MS method was studied. Three soymilk samples were spiked with authentic isoflavones at 20.0 mg/L and analyzed. Recoveries were found to be in the range of 97.5 ± 0.7% and 102.0 ± 1.2%.

In conclusion, baicalin functionalized core-shell superparamagnetic nanoparticles (BMNPs) of high water suspensibility were successfully prepared. The synthetic method is easy to carry out, and the amount of baicalin loaded to the MNPs is larger than previous one³³. Using BMNPs as magnetic solid phase extract sorbent, a selective extraction of isoflavones from soymilk was developed, and the extracted isoflavones were subsequently subjected to HPLC–ESI-MS/MS analysis. This is the first time that BMNPs was used in quantitative analysis. As demonstrated in this work, the proposed MSPE method is effective, easy to carry out, and applicable to fast HPLC-MS/MS quantification of isoflavones in soymilk and other liquid soybean products, such as soy beverages.

• 1.Setchell KD, Faughnan MS, Avades T, Zimmer-Nechemias L, Brown NM, Wolfe BE, Brashear WT, Desai P, Oldfield MF, Botting NP, Cassidy A. Comparing the pharmacokinetics of daidzein and genistein with the use of 13C-labeled tracers in premenopausal women. Am J Clin Nutr. 2003;77:411–419. doi: 10.1093/ajcn/77.2.411. [DOI] [PubMed] [Google Scholar]
• 2.Franke AA, Custer LJ, Cerna CM, Narala KK. Quantitation of phytoestrogens in Legumes by HPLC. J Agric Food Chem. 1994;42:1905–1913. [Google Scholar]
• 3.Murphy PA, Song T, Buseman G, Barua K, Beecher GR, Trainer D, Holden J. Isoflavones in retail and institutional soy foods. J Agric Food Chem. 1999;47:2697–2704. doi: 10.1021/jf981144o. [DOI] [PubMed] [Google Scholar]
• 4.Nagata C, Takatsuka N, Kawakami N, Shimizu H. Soy product intake and hot flashes in japanese women: results from a community-based prospective study. Am J Epidemiol. 2001;153:790–793. doi: 10.1093/aje/153.8.790. [DOI] [PubMed] [Google Scholar]
• 5.Alekel DL, Germain AS, Peterson CT, Hanson KB, Stewart JW, Toda T. Isoflavone-rich soy protein isolate attenuates bone loss in the lumbar spine of perimenopausal women. Am J Clin Nutr. 2000;72:844–852. doi: 10.1093/ajcn/72.3.844. [DOI] [PubMed] [Google Scholar]
• 6.Reinwald S, Weaver CM. Soy isoflavones and bone health: a double-edged sword?⊥. J Nat Prod. 2005;69:450–459. doi: 10.1021/np058104g. [DOI] [PubMed] [Google Scholar]
• 7.Goodman-Gruen D, Kritz-Silverstein D. Usual dietary isoflavone intake is associated with cardiovascular disease risk factors in postmenopausal women. J Nutr. 2001;131:1202–1206. doi: 10.1093/jn/131.4.1202. [DOI] [PubMed] [Google Scholar]
• 8.Taku K, Umegaki K, Sato Y, Taki Y, Endoh K, Watanabe S. Soy isoflavones lower serum total and LDL cholesterol in humans: a meta-analysis of 11 randomized controlled trials. Am J Clin Nutr. 2007;85:1148–1156. doi: 10.1093/ajcn/85.4.1148. [DOI] [PubMed] [Google Scholar]
• 9.Setchell KD, Brown NM, Zhao X, Lindley SL, Heubi JE, King EC, Messina MJ. Soy isoflavone phase II metabolism differs between rodents and humans: implications for the effect on breast cancer risk. Am J Clin Nutr. 2011;94:1284–1294. doi: 10.3945/ajcn.111.019638. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 10.Sakamoto T, Horiguchi H, Oguma E, Kayama F. Effects of diverse dietary phytoestrogens on cell growth, cell cycle and apoptosis in estrogen-receptor-positive breast cancer cells. J Nutr Biochem. 2010;21:856–864. doi: 10.1016/j.jnutbio.2009.06.010. [DOI] [PubMed] [Google Scholar]
• 11.Cappelletti V, Fioravanti L, Miodini P, Di Fronzo G. Genistein blocks breast cancer cells in the G2M phase of the cell cycle. J Cell Biochem. 2000;79:594–600. [PubMed] [Google Scholar]
• 12.Barnes S. The biochemistry, chemistry and physiology of the isoflavones in soybeans and their food products. Lymph Res Biol. 2010;8:89–98. doi: 10.1089/lrb.2009.0030. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 13.Messina M, Messina V, Jenkins DJA. Can breast cancer patients use soyafoods to help reduce risk of CHD? Brit J Nutr. 2012;108:810–819. doi: 10.1017/S0007114512001900. [DOI] [PubMed] [Google Scholar]
• 14.Coward L, Barnes NC, Setchell KDR, Barnes S. Genistein, daidzein, and their .beta.-glycoside conjugates: antitumor isoflavones in soybean foods from American and Asian diets. J Agric Food Chem. 1993;41:1961–1967. [Google Scholar]
• 15.Messina M. Modern applications for an ancient bean: soybeans and the prevention and treatment of chronic disease. J Nutr. 1995;125:567S–569S. doi: 10.1093/jn/125.3_Suppl.567S. [DOI] [PubMed] [Google Scholar]
• 16.Klejdus B, Vacek J, Beneova L, Kopecky J, Lapik O, Kuba V. Rapid-resolution HPLC with spectrometric detection for the determination and identification of isoflavones in soy preparations and plant extracts. Anal Bioanal Chem. 2007;389:2277–2285. doi: 10.1007/s00216-007-1606-3. [DOI] [PubMed] [Google Scholar]
• 17.Klump SP, Allred MC, MacDonald JL, Ballam JM. Determination of isoflavones in soy and selected foods containing soy by extraction, saponification, and liquid chromatography: Collaborative Study. J AOAC Int. 2001;84:1865–1883. [PubMed] [Google Scholar]
• 18.Otieno DO, Rose H, Shah NP. Profiling and quantification of isoflavones in soymilk from soy protein isolate using extracted ion chromatography and positive ion fragmentation techniques. Food Chem. 2007;105:1642–1651. [Google Scholar]
• 19.Peng Y, Chu Q, Liu F, Ye J. Determination of isoflavones in soy products by capillary electrophoresis with electrochemical detection. Food Chem. 2004;87:135–139. doi: 10.1021/jf030280c. [DOI] [PubMed] [Google Scholar]
• 20.Chung I-M, Kim E-H, Kim S-L, Kim S-H. Comparison of isoflavones and anthocyanins in soybean [Glycine max (L.) Merrill] seeds of different seeding dates. J Agric Food Chem. 2012;60:10196–10202. doi: 10.1021/jf3031259. [DOI] [PubMed] [Google Scholar]
• 21.Lin L-z, Harnly JM. Quantitation of flavanols, proanthocyanidins, isoflavones, flavanones, dihydrochalcones, stilbenes, benzoic acid derivatives using UV absorbance after identification by LC-MS. J Agric Food Chem. 2012;60:5832–5840. doi: 10.1021/jf3006905. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 22.Luthria DL, Biswas R, Natarajan S. Comparison of extraction solvents and techniques used for the assay of isoflavones from soybean. Food Chem. 2007;105:325–333. [Google Scholar]
• 23.Murphy PA. Separation of genistin, daidzin and their aglucones, and coumesterol by gradient high-performance liquid chromatography. J Chromatogr A. 1981;211:166–169. [Google Scholar]
• 24.Rostagno MA, Palma M, Barroso CG. Ultrasound-assisted extraction of soy isoflavones. J Chromatogr A. 2003;1012:119–128. doi: 10.1016/s0021-9673(03)01184-1. [DOI] [PubMed] [Google Scholar]
• 25.Rostagno MA, Palma M, Barroso CG. Pressurized liquid extraction of isoflavones from soybeans. Anal Chim Acta. 2004;522:169–177. [Google Scholar]
• 26.Araújo JMA, Silva MV, Chaves JBP. Supercritical fluid extraction of daidzein and genistein isoflavones from soybean hypocotyl after hydrolysis with endogenous β-glucosidases. Food Chem. 2007;105:266–272. [Google Scholar]
• 27.Yang F, Ma Y, Ito Y. Separation and purification of isoflavones from a crude soybean extract by high-speed counter-current chromatography. J Chromatogr A. 2001;928:163–170. doi: 10.1016/s0021-9673(01)01144-x. [DOI] [PubMed] [Google Scholar]
• 28.Careri M, Corradini C, Elviri L, Mangia A. Optimization of a rapid microwave assisted extraction method for the liquid chromatography–electrospray-tandem mass spectrometry determination of isoflavonoid aglycones in soybeans. J Chromatogr A. 2007;1152:274–279. doi: 10.1016/j.chroma.2007.03.112. [DOI] [PubMed] [Google Scholar]
• 29.Rostagno MA, Palma M, Barroso CG. Solid-phase extraction of soy isoflavones. J Chromatogr A. 2005;1076:110–117. doi: 10.1016/j.chroma.2005.04.045. [DOI] [PubMed] [Google Scholar]
• 30.Rostagno MA, Villares A, Guillamón E, García-Lafuente A, Martínez JA. Sample preparation for the analysis of isoflavones from soybeans and soy foods. J Chromatogr A. 2009;1216:2–29. doi: 10.1016/j.chroma.2008.11.035. [DOI] [PubMed] [Google Scholar]
• 31.Wiseman H, Casey K, Clarke DB, Barnes KA, Bowey E. Isoflavone aglycon and glucoconjugate content of high-and low-soy u.k. foods used in nutritional studies. J Agric Food Chem. 2002;50:1404–1410. doi: 10.1021/jf011243t. [DOI] [PubMed] [Google Scholar]
• 32.Toro-Funes N, Odriozola-Serrano I, Bosch-Fusté J, Latorre-Moratalla ML, Veciana-Nogués MT, Izquierdo-Pulido M, Vidal-Carou MC. Fast simultaneous determination of free and conjugated isoflavones in soy milk by UHPLC–UV. Food Chem. 2012;135:2832–2838. doi: 10.1016/j.foodchem.2012.06.011. [DOI] [PubMed] [Google Scholar]
• 33.Qing L-S, Xiong J, Xue Y, Liu Y-M, Guang B, Ding L-S, Liao X. Using baicalin-functionalized magnetic nanoparticles for selectively extracting flavonoids from Rosa chinensis. J Sep Sci. 2011;34:3240–3245. doi: 10.1002/jssc.201100578. [DOI] [PubMed] [Google Scholar]
• 34.Lin P-C, Tseng M-C, Su A-K, Chen Y-J, Lin C-C. Functionalized magnetic nanoparticles for small-molecule isolation, identification, and quantification. Anal Chem. 2007;79:3401–3408. doi: 10.1021/ac070195u. [DOI] [PubMed] [Google Scholar]
• 35.Song Y, Zhao S, Tchounwou P, Liu Y-M. A nanoparticle-based solid-phase extraction method for liquid chromatography-electrospray ionization-tandem mass spectrometric analysis. J Chromatogr A. 2007;1166:79–84. doi: 10.1016/j.chroma.2007.07.074. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 36.Gao Q, Luo D, Bai M, Chen Z-W, Feng Y-Q. Rapid determination of estrogens in milk samples based on magnetite nanoparticles/polypyrrole magnetic solid-phase extraction coupled with liquid chromatography–tandem mass spectrometry. J Agric Food Chem. 2011;59:8543–8549. doi: 10.1021/jf201372r. [DOI] [PubMed] [Google Scholar]
• 37.Qing L-S, Xue Y, Deng W-L, Liao X, Xu X-M, Li B-G, Liu Y-M. Ligand fishing with functionalized magnetic nanoparticles coupled with mass spectrometry for herbal medicine analysis. Anal Bioanal Chem. 2011;399:1223–1231. doi: 10.1007/s00216-010-4399-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 38.Wang L, Bao J, Wang L, Zhang F, Li Y. One-pot synthesis and bioapplication of amine-functionalized magnetite nanoparticles and hollow nanospheres. Chem-Eur J. 2006;12:6341–6347. doi: 10.1002/chem.200501334. [DOI] [PubMed] [Google Scholar]
• 39.Rostagno MA, Palma M, Barroso CG. Solid-phase extraction of soy isoflavones. J Chromatogr A. 2005;1076:110–117. doi: 10.1016/j.chroma.2005.04.045. [DOI] [PubMed] [Google Scholar]
• 40.Zafra-Gómez A, Garballo A, García-Ayuso LE, Morales JC. Improved sample treatment and chromatographic method for the determination of isoflavones in supplemented foods. Food Chem. 2010;123:872–877. [Google Scholar]
• 41.Matsumoto D, Kotani A, Hakamata H, Takahashi K, Kusu F. Column switching high-performance liquid chromatography with two channels electrochemical detection for high-sensitive determination of isoflavones. J Chromatogr A. 2010;1217:2986–2989. doi: 10.1016/j.chroma.2010.02.050. [DOI] [PubMed] [Google Scholar]