LC/UV/ESI-MS Analysis of Isoflavones in Edamame and Tofu Soybeans

Abstract

High-performance liquid chromatography coupled with ultraviolet and electrospray ionization mass spectrometry (HPLC/UV/ESI-MSD) was applied to the study of isoflavones in both Edamame and Tofu soy varieties, from which the immature fresh soybeans or the mature soybean seeds are consumed, respectively. Positive atmospheric pressure interface (API) MS and MS/MS were used to provide molecular mass information and led to the identification of a total 16 isoflavones, including three aglycones, three glycosides, two glycoside acetates, and eight glycoside malonates. The major isoflavones in soybean seeds were daidzein and genistein glycoside and their malonate conjugates. Trace levels of daidzein and genistein acetyl glycosides were found only in the mature dry soybean seeds. To facilitate quantitative analysis, acid hydrolysis during extraction of soy samples was selected to convert the various phytoestrogen conjugates into their respective isoflavone aglycones, allowing accurate quantitation of total phytoestrogens as aglycones. On the basis of HPLC combined with UV and MS detection, all three targeted soy isoflavone aglycones, daidzein, genistein and glycitein in hydrolyzed extracts were successfully quantified within 25 min with formononetin used as the internal standard. The standard curves of UV detection were fitted in the range of 14.16-29000 ng/mL for daidzein, 15.38-31500 ng/mL for genistein, and 11.72-24000 ng/mL for glycitein. For MS detection, the standard curves were established in the range of 3.54-1812.5 ng/mL for daidzein, 3.85-1968.75 ng/mL for genistein, and 2.93-1500 ng/mL for glycitein. Good linearities (r(2) > 0.999 for UV and r(2) > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and precision (RSD) were within 10% for UV detection and 15% for MS detection (n = 10). Using this method, the phytoestrogen levels of total isoflavone aglycones from 30 soybean seed varieties were then evaluated for confirmation of the technique. Total isoflavones ranged across the varieties from 0.02 to 0.12% in the Edamame varieties, which are harvested while the seeds are still immature, and from 0.16 to 0.25% in Tofu varieties, harvested when the seeds are physiologically mature. While the literature has focused on the isoflavone content of soy products and processing soy, this report provides a reliable analytical technique for screening of authenticated fresh immature Edamame soybeans and Tofu soybeans.

Bioactive Phenolic Compounds of Soybean (Glycine max cv. Merit): Modifications by Different Microbiological Fermentations

“…Wu et al [2004] have also reported the presence of different isomers of malonylglycosides of genistein, daidzein and glycitein, that were the most common in soybeans seeds. Therefore, the presence of genistein malonylglycoside and genistein acetylglycoside in the control soybean was in agreement with that reported by the mentioned authors.…”

“…It has been observed that the content of isofl avones in soybean showed a large range of concentrations depending on several factors, mainly cultivation, the degree of maturation, and the environmental culture conditions, as well as the variety and origin. Various studies have reported the presence of malonyl and acetyl glycosides of daidzein, genistein and glycitein, in the raw soybean seed in diverse concentrations, mainly in raw mature seeds, but mainly the presence of the glycosides was highlighted [Rostagno et al, 2004;Wu et al, 2004].…”

Vegetable soybean: seed composition and production research

“…Vegetable soybeans contain about 50 percent more isoflavones than mature soybean seeds (Koes et al, 2005;Wu et al, 2004). The content of isoflavones was 78 to 220 µg/g dried seed) and tocopherols (vitamin E) ranged from 84 to 128 µg/g dry seed (Mohamed et al, 2001), while the contents of the individual isoflavones were reported to differ depending on soybean variety and cultivation conditions (Lee et al, 2003).…”

Food and Dietary Supplement Package Labeling—Guidance from FDA's Warning Letters and Title 21 of the Code of Federal Regulations

“…314 Please note that "edamame" is a botanical term, and not a trade name or a fanciful name. [315][316][317] In other warning letters, data from an FDA inspection of the manufacturing facility were used to determine that the label failed to disclose various allergens.…”