Barnes S, Coward L, Kirk M, Sfakianos J
Department of Biochemistry & Molecular Genetics, University of Alabama at Birmingham, 35294, USA.
Proc Soc Exp Biol Med. 1998 Mar;217(3):254-62. doi: 10.3181/00379727-217-44230.
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.
目前人们对膳食异黄酮，尤其是大豆异黄酮染料木黄酮在降低多种慢性疾病风险方面的作用颇为关注，这就需要对食品基质、各种生理体液和组织中的异黄酮及其代谢产物进行快速、灵敏且精确的分析测定。与基于气相色谱（GC）的方法相比，高效液相色谱（HPLC）的优势在于，所有结合型和非结合型异黄酮及其代谢产物无需衍生化即可分离和分析。质谱分析的一项重要进展是在HPLC和质谱仪之间引入了有效的接口，即电喷雾电离（ESI）接口和加热雾化器-大气压化学电离（HN-APCI）接口。由于胆汁或尿液等体液中的异黄酮浓度，对于GC-MS和许多其他分析方法至关重要的初步萃取在这里并非必要。这立即解决了寻找有效固相萃取程序这一棘手问题。使用反相HPLC-ESI-MS，有可能在单次20分钟的分析中获得所有异黄酮代谢产物的质量/强度图谱。血清/血浆样品中异黄酮结合物的分析需要先进行萃取，但结合物可通过毛细管反相HPLC-ESI-MS或在常规反相柱上通过HPLC-HN-APCI-MS完整测定。在这两种情况下，通过使母体异黄酮分子离子在三重四极杆质谱仪中发生碰撞诱导解离以形成特定子离子来实现特异性。当仅需测定血液中的总异黄酮及其代谢产物时，可直接在血清/血浆样品中进行水解，然后通过乙醚或乙酸乙酯溶剂萃取回收异黄酮。异黄酮苷元可在等度溶剂条件下通过HPLC-MS进行分析，从而大幅缩短分析时间并为自动化开辟前景。因此，HPLC-MS是一种广泛适用于植物雌激素研究中主要问题的技术。
