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SECOND INTERNATIONAL SYMPOSIUM
ON THE ROLE OF SOY
IN PREVENTING AND TREATING CHRONIC DISEASESeptember 15-18, 1996
Brussells, Belgium
POSTER ABSTRACTSAnalysis of isoflavones and their Meta-bolites in Plasma by HPLC-Mass Spectrometry.
Coward, M. Kirk and S. Barnes
Department of Pharmacology and Toxicology and the Mass Spectrometry Shared Facility, Comprehensive Cancer Center, University of Alabama at Birmingham, AL 35294, USA.
Due to the current interest in soybean isoflavones and the prevention of several chronic diseases, both animal and clinical studies are on the rise. This has lead to the need for rapid, economic, and quantitative assays to determine the concentration of isoflavones and their metabolites in plasma. Previous methods require time consuming sample preparation or lack the sensitivity to determine trace levels of the isoflavones. We have developed methods using HPLC-heated nebulizer-atmospheric pressure chemical ionization mass spectrometry (HPLC-HN-APCI-MS) and HPLC electrospray ionization mass spectrometry (HPLC- HN- ESI- MS) to obtain quantitative data at low nM concentrations. Plasma isoflavones are present mostly as their sulfate and glucuronide conjugates. To measure the total concentration of genistein, daidzein, desmethylangolensin, dihydrodaidzein and equol in plasma, their conjugates are hydrolyzed enzymatically and the aglucones are extracted into ether. The ether extract is evaporated, redissolved in aqueous methanol and an aliquot is analyzed by reversed-phase HPLC-HN-APCI-MS (Clin Chim Acta 247: 121-142, 1996). The isoflavones and metabolites are eluted isocratically with 40% acetonitrile. Specifically for the isoflavones is obtained using multiple reaction ion monitoring (MRM). Reproducible and quantitative results can be obtained for concentrations as low as 5 nM in 1 ml of plasma. While total isoflavone concentrations are adequate for most purposes, the concentrations of the sulfate and glucuronide conjugates and the concentrations can be obtained by selective enzyme hydrolysis. Plasma is extracted before hydrolysis, or after hydrolysis using an enzyme specific for elther sulfates or glucuronides. The aglucones are extracted and measured as described above. For direct analysis of the intact conjugates, plasma is extracted with a Sep-Pak cartridge, and the conjugates analyzed by reversed-phase HPLC-ESI-MS. This method allows for the detection of multiple conjugates (disulfates, sulfate-glucuronides, diglucuronides), but due to the unavailability of standards currently lacks the ability to give quantatative values for these conjugates. In summary, serveral HPLC-MS methods are available for the specific quantitative measurement of plasma isoflavones and their metabolites.
Supported by a grant from the United Soybean Board.
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