HPLC-Mass Spectrometry Analysis of Isoflavones

The current interest in the role of dietary isoflavonoids, particularly the soy isoflavone genistein, in lowering the risk of several chronic diseases, has led to the need for rapid, sensitive and precise assays for isoflavones and their metabolites in food matrices and in various physiological fluids and tissues. HPLC has the advantage over GC-based methods in that all the conjugated and unconjugated isoflavonoids and their metabolites can be separated and analyzed without the need for derivatization. An important advance in mass spectrometry has been the introduction of effective interfaces between the HPLC and the mass spectrometer, namely the electrospray ionization (ESI) and the heated nebulizer-atmospheric pressure chemical ionization (HN-APCI) interfaces. Because of the isoflavonoid concentrations in fluids such as bile or urine, preliminary extraction, so essential for GC-MS and many other analytical methods, is not necessary. This immediately overcomes the thorny issue of finding an effective solid-phase extraction procedure. Using reversed-phase HPLC-ESI-MS, it is possible to obtain a mass/intensity map of all isoflavonoid metabolites in a single 20 min analysis. Analysis of isoflavonoid conjugates in serum/plasma samples requires initial extraction, but the conjugates can be measured intact either by capillary reversed-phase HPLC-ESI-MS or on regular reversed-phase columns by HPLC-HN-APCI-MS. In both cases, specificity is obtained by causing the parent isoflavonoid molecular ions to undergo collision-induced dissociation to form specific daughter ions in a triple quadrupole MS instrument. When it is only necessary to measure the total isoflavonoids and their metabolites in blood, hydrolysis can be performed directly in serum/plasma samples and isoflavonoids recovered by ether or ethyl acetate solvent extraction. The isoflavone aglucones can be analyzed by HPLC-MS under isocratic solvent conditions, thereby drastically shortening analysis time and opening up prospects for automation. Therefore, HPLC-MS is a technique that is broadly applicable to the major issues in phytoestrogen research.