Separation and Isolation of (S)-Equol: The Biologically Relevant Metabolite of the Isoflavone Daidzein
high performance liquid chromatography
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Publisher:The Ohio State University
Series/Report no.:The Ohio State University. Department of Pharmaceutical Sciences Honors Theses; 2006
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.
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