Separation and Isolation of (S)-Equol: The Biologically Relevant Metabolite of the Isoflavone Daidzein
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Date
2006-06
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Publisher
The Ohio State University
Abstract
Isoflavones, a class of phytochemicals abundant in soy, have recently been investigated for their biological activity in humans. One of the primary soy isoflavones, daidzein, is transformed into S(-)-equol, a metabolite produced by microflora in the human intestine. Synthesized equol is currently available as a racemic mixture, while only the biologically active S(-)-equol is created by bacteria in humans. Because of the interest in this compound, a need exists for isolating and identifying the S(-) isomer for further analytical and biological studies. The objective of this study is to create a simple, accurate, and cost effective High Performance Liquid Chromatography (HPLC) method for separating the two enantiomers of equol using easily obtainable compounds. (+/-)-Equol was purchased and reacted with methoxytriflurophenyl acyl chloride to produce diastereomers. The diastereomers were analyzed for purity by HPLC. Successful derivatization of the equol-MTPA esters was confirmed by HPLC-Mass Spectrometry (MS). The two diastereomers were separated via HPLC on a Luna Silica column (4.6 mm x 150 mm, 3 μm packing), 1 mL/min flow at 25°C, gradient = 1/99 MtBE:hexane to 4.3/96.7 MtBE:hexane over 49.5 min, and separation was confirmed with HPLC-MS. Next, fractions will be collected of each diastereomer, the MTPA will be removed, and Circular Dichroism Spectra analysis will be performed on both collections to ensure separation has been achieved.
Description
First Place, CFAES Forum, 2005
Keywords
equol, chiral, chiral derivatization, isoflavone, high performance liquid chromatography, chiral separation, metabolite