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.

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 1.0-4320.0 nM using 75 microL of rat plasma. Rat plasma samples were hydrolyzed by adding an enzyme mixture from Helix pomatia containing glucuronidase and sulfatase to convert the isoflavone beta-glycosides daidzin, genistin, and glycitin to their active aglycone forms. A liquid-liquid extraction method using ethyl acetate as the extraction solvent was used to extract aglycones and the internal standards (phenolphthalein beta-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 water-acetonitrile (85 + 15), and injected into a Zorbax SB-CN reversed-phase column (4.6 x 75 mm, 3.5 microm 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 <2.6% relative standard deviation and <10% relative error, respectively. Recovery of the reported isoflavones with this method varied from 86 to 100%.