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To study the safety and potential health benefits of soy isoflavones, a rapid and simple method based on liquid chromatography combined with mass spectrometry (LC/MS) and photodiode array detector (PDA) was developed for the determination of isoflavones in rat plasma. The analytes included daidzein, genistein, glycitein, equol, 4-ethyl phenol, and biochanin A over a concentration range of 1.04320.0 nM using 75 L of rat plasma. Rat plasma samples were hydrolyzed by adding an enzyme mixture from Helix pomatia containing glucuronidase and sulfatase to convert the isoflavone -glycosides daidzin, genistin, and glycitin to their active aglycone forms. A liquidliquid extraction method using ethyl acetate as the extraction solvent was used to extract aglycones and the internal standards (phenolphthalein -d glucuronide, 4-methylumbelliferyl sulfate, and apigenin) from digested plasma samples. The extract was evaporated to dryness under a nitrogen stream, reconstituted with 0.1%formic acid in wateracetonitrile (85 + 15), and injected into a Zorbax SB-CN reversed-phase column (4.6 75 mm, 3.5 m particle size). The Micromass ZQ detector was operated in the positive ion selected-ion monitoring mode. The flow rate for LC was 1.0 mL/ min, with a split where 25% of the effluent was introduced into the electrospray ionization probe of the MS instrument and 75% into the PDA. The chromatographic run time was 16.0 min, with delay of 10 min/injection. The interday precision and accuracy of the standard samples were &amp;lt;2.6% relative standard deviation and &amp;lt;10% relative error, respectively. Recovery of the reported isoflavones with this method varied from 86 to 100%.

journal of aoac international

An Accurate and Reproducible Method for the Quantitative Analysis of Isoflavones and Their Metabolites in Rat Plasma Using Liquid Chromatography/Mass Spectrometry Combined with Photodiode Array Detection