Simultaneous Identification of Soyasaponins and Isoflavones and Quantification of Soyasaponin B b in Soy Products, Using Liquid Chromatography/Electrospray Ionization-Mass Spectrometry (original) (raw)
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Microchemical Journal, 2013
This article reports the development of a modified approach of the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) methodology for the extraction of naturally occurring substances from solid soy-based products. The use of this methodology offers advantages such as simplicity and ease of use, especially taking into account the complexity of food matrices. Here, capillary electrophoresis coupled with electrospray ionization mass spectrometry (CE-ESI-MS) was applied for the separation and quantification of isoflavones in soy products. Because of the importance of the different forms of isoflavones as regards their biological activity, a method that would permit both aglycones and the glycoside forms of isoflavones to be determined was developed. The proposed method was successfully applied to the analysis of the glycosylated forms glycitin, daidzin and genistin, and the aglycones formononetin, biochanin A, glycitein, daidzein, and genistein in commercially available soy biscuit samples. The parameters evaluated for the validation of the method developed included the linearity of the calibration plots, the detection and quantification limits, the existence of matrix effect(s), repeatability, reproducibility and recovery. The results of the validation process revealed that the proposed method is a possible alternative in routine analysis of isoflavones in food matrices.
Food Chemistry, 2007
Dietary supplements on soy based foods and beverages are increasingly gaining prominence all over the world. In this study, liquid chromatography coupled with positive electrospray ionisation tandem mass spectrometry (LC-ESI-MS) and diode array detection was used for the quantitation and characterisation of isoflavones in fermented and unfermented soymilk made from soy protein isolate SUPRO 590. Bifidobacterium animalis ssp. lactis Bb12 was used for the fermentation of soymilk. The isoflavones were found to produce characteristic radical ions as well as molecules of H2O, CO2, a sugar unit, and an alcohol through collision-induced fragmentation. Product ion fragments revealed unique fragmentation pathways for each isoflavone compound. Characteristic fragmentation of different isoflavones were unequivocally identified and differentiated. The occurrence of aldehydes such as pentanal, ethanal and methanal was shown to be specifically linked with isoflavone aglycones, daidzein, genistein and glycitein, respectively. Main glycosides such as genistin, daidzin and glycitin as well as the acetyl-, and malonyl forms also showed respective aglycone ions in their spectra fragmentation. Thus positive ion fragmentation was important in the absolute confirmation of isoflavones and to reveal the occurrence of other related compounds such as aldehydes in soymilk.
Quantification of the Group B Soyasaponins by High-Performance Liquid Chromatography
Journal of Agricultural and Food Chemistry, 2002
High-performance liquid chromatographic methods were developed for the isolation and quantitative determination of the group B soyasaponins, including 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated soyasaponins Rg, g, and a, and their non-DDMP counterparts, soyasaponins V, I, and II, respectively, with formononetin used as the internal standard. The limits of quantification for soy products were 0.11-4.86 µmol/g. The within-day and between-days assay coefficients of variation were <9.8 and < 14.3%, respectively. The group B soyasaponin concentrations in 46 soybean varieties ranged from 2.50 to 5.85 µmol/g. Soy ingredients (soybean flour, toasted soy hypocotyls, soy protein isolates, textured vegetable protein, soy protein concentrates, and Novasoy) and soy foods (commercial soy milk, tofu, and tempeh) contained the group B soyasaponins from 0.20 to 114.02 µmol/g. There was no apparent correlation between isoflavone and soyasaponin concentrations in the soy products examined.
Identification of soyasaponins by liquid chromatography–thermospray mass spectrometry
Journal of Chromatography A, 1997
Saponins are important bioactive molecules widespread in the plant kingdom. Soyasaponins, isolated from Glicine max (Leguminosae), have been shown to exhibit various biological activities, e.g., an inhibitory effect on lipid-oxidation and liver-lesion generation and an improving effect on hyper-cholesteremia. Mass spectral investigation of these metabolites requires soft ionization techniques such as desorption chemical ionization, fast atom bombardment and thermospray mass spectrometry. A high-performance liquid chromatographic method was developed for thermospray mass spectrometric analysis of saponins contained in soybean flour extracts. The analyses were performed using a ternary eluent (wateracetonitrile-methanol) in gradient conditions with post-column addition of aqueous ammonium acetate. Six saponin components could be separated and identified. The mass spectra obtained provided information concerning both molecular masses and aglycone composition.
Journal of Agricultural and Food Chemistry, 2005
Soyasaponins are phytochemicals of major interest for health. Their identification and quantification remain difficult owing to the large number of structural isomers in soybeans and the lack of stable standards. In this study, a rapid method using high performance liquid chromatography (HPLC) using a UV detector (205 nm) was developed to identify and quantify soyasaponins belonging to group B and compare them with isoflavones in different soy materials. 2,3-Dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated soyasaponins were determined using external calibration or a molecular mass ratio after alkaline hydrolysis to cleave their DDMP moieties. The detection limit of soyasaponin I, used as a reference molecule to simplify the analysis, was 0.065 µmol/g. Soyasaponin contents in seven soybean varieties ranged from 13.20 to 42.40 µmol/g in the germ and from 2.76 to 6.43 µmol/g in the cotyledons. The within-day and between-days variation coefficients did not exceed 7.9 and 9.0%, respectively, for the major soyasaponins. Soyasaponin B quantification in different soy-based health supplements was reported along with measurements of their isoflavone content to provide information on the variability of these bioactive compounds among different types of soy food materials.
Analytica Chimica Acta, 2012
Capillary zone electrophoresis coupled with electrospray ionization mass spectrometry (CZE-ESI-MS) has been applied for the first time for the separation and quantification of isoflavones in soy products. The proposed method was successfully applied to the determination of seven isoflavones, including aglycones and glucosides, in soy drink. The target compounds were the glucosides daidzin and genistin, and the aglycones daidzein, genistein, formononetin, biochanin A and glycitein. During CE separation in positive mode, the analytes were present as anions, and MS detection was carried out in ESI positive-ion mode. To prevent the frequent drops in current and to improve the resolution in the separation of analytes in anionic form, a programmed nebulizing gas pressure (PNP) was applied along the analysis. Extraction of isoflavones from soy drinks was carried out by liquid-liquid extraction using ethanol. The proposed extraction procedure is simple, efficient, and affords reproducible results. Quantification of the isoflavones in soy drinks using the external standard method did not produce good results; therefore, both internal standard and standard addition quantification methods were used, obtaining significantly similar results. The detection limits found were lower than 3.2 g L −1 .
Journal of Agricultural and Food Chemistry, 1994
Identification of isoflavone glycosidic conjugates from several soy products was carried out by HPLCmass spectrometry. Positive ion mass spectra obtained using the heated nebulizer-atmospheric pressure chemical ionization interface gave the most sensitive and structurally useful information about each isoflavone conjugate. Although extraction of isoflavones from soy products with 80% aqueous methanol at room temperature was just as efficient as at 60-80 "C, extraction at higher temperatures caused changes in isoflavone composition and should be avoided. Soybeans and defatted soy flour (which had been minimally heated during their preparation) contained mostly isoflavone 6"-O-malonylglucoside conjugates, with lesser quantities of the @-glucosides and only trace amounts of 6"-0-acetylglucoside conjugates. Soy milk, tofu, and soy molasses, each of which involves heating to 100 "C during their manufacture, contained mostly isoflavone @-glucosides. Toasted soy flour and an isolated soy protein had moderate amounts of each of the isoflavone conjugates.
Biological Mass Spectrometry, 1989
The thermospray mass spectra of the phytoestrogens have intense protonated molecular ions but contain few or no ions indicative of structure. Tandem mass spectrometry (MS/MS) was used to obtain daughter ion spectra containing ions unique to the different structural characteristics of each phytoestrogen subclass and was used both to coofirm identification and propose structures for unknowns. In addition to unique daughter ion spectra, MS/MS was used as a class identifier to detect phytoestrogens through the neutral loss of 56 (due to consecutive losses of CO) that is common to all members of this family. Several sources of soy protein were investigated to confirm the presence or absence of phytoestrogens. In one preparation investigated, daidzein and genistein were detected as well as an unknown phytaestrogen of the Biochanin A subclass. This unknown has been tentatively identified as 6,7dihydroxy-4'-methoxyisoflavone using its daughter ion spectrum.
Analytical and Bioanalytical Chemistry, 2007
A rapid-resolution HPLC/UV-VIS DAD separation method (which takes <1 min) for the determination and identification of genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin, formononetin, sissotrin and biochanin A in fmol quantities in submicroliter sample volumes was optimized. A linear gradient elution (0 min 22% B, 1.0 min 80% B, 1.4 min 100% B, 1.8 min 22% B) using a mobile phase containing 0.2 % (v/v) acetic acid (solvent A) and methanol (solvent B) was applied on a Zorbax SB C 18 column (1.8 μm particle size) at 80°C. The method was verified using samples of bits of soy and methanolic extracts from Trifolium pratense, Iresine herbstii and Ononis spinosa plants. Pseudobaptigenin glucoside, irilone, prunetin, texasin, tlatlancuayin and other isoflavones, in addition to aglycones of isoflavones and their β-glucosides and malonyl and acetyl derivatives, were identified by UV-VIS DAD and electrospray mass spectrometric (ESI-MS) detection in the extracts.