**3. Isolation**

The isoflavonoid aglycones such as genistein and daidzein are compounds with a low polar‐ ity and hence practically insoluble in water. The polarity is lowered by methylation, as in formononetin and biochanin A. After glycosylation, the water solubility increases; glucosides have a higher solubility in water than their malonylated and acetylated derivatives. The glycosidic bond may be hydrolyzed under acidic or basic conditions [15]. Early extraction of isoflavones was performed by refluxing alcohol, but had the disadvantage of converting malonyl‐ and acetyl‐glucosides into glucosides and aglycones [16]. A mixture of methanol 80% was as well proposed [17]. An optimized extraction method was developed by Griffith and Collison, using acetonitrile/water, without the addition of an acid [18]. Acetonitrile is considered to yield higher extraction ratios than solvents such as acetone, ethanol and methanol, during the analysis of 12 main soy isoflavones from foods; the organic solvent (53%) is mixed with water [19]. The preparative isolation of isoflavones could be achieved by high‐speed countercurrent chromatography (HSCCC). In one setting of HSCCC, acid‐ free solvents were employed; the isolation of malonylglucosides was performed with the aid of a solvent *tert*‐butyl‐methyl ether/*n*‐butanol/acetonitrile/water in a ratio of 1/3/1/5 [20]. Monoglucosylated and acetylated isoflavones were obtained more recently by HSCCC after a cleaning‐up step on Amberlite XAD‐7 material [21].

Quantification of isoflavones is usually performed by HPLC‐DAD, using reversed‐phase columns and eluents containing 95% acetonitrile with 0.1% trifluoroacetic acid. Validated methods with good peak resolution are available [18]. Detection may be performed at 262 nm. For the quanti‐ fication in biologic samples (urine, saliva and blood), HPLC‐MS/MS spectrometry is employed after solid‐phase extraction (SPE) of isoflavones; the glycosides are hydrolyzed enzymatically prior to SPE [22].
