Department of Analytical Chemistry, Silesian University of Technology, 7 M. Strzody Str., 44-100 Gliwice, Poland.
Anal Bioanal Chem. 2011 Mar;399(9):3211-9. doi: 10.1007/s00216-010-4206-6. Epub 2010 Oct 9.
A rapid, ultra high-performance liquid chromatographic (UHPLC) method has been developed and validated for simultaneous identification and analysis of the isoflavones genistein, daidzein, glycitin, puerarin, and biochanin A, and the flavonoids (±)-catechin, (-)-epicatechin, rutin, hesperidin, neohesperidin, quercitrin, and hesperetin in human urine. Urine samples were incubated with β-glucuronidase/sulfatase. UHPLC was performed with a Hypersil Gold (50 × 2.1 mm, 1.9 μm) analytical column. Elution was with a gradient prepared from aqueous trifluoroacetic acid (0.05%) and acetonitrile. UV detection was performed at 254 and 280 nm. The calibration curves were indicative of good linearity (r(2) ≥ 0.9992) in the range of interest for each analyte. LODs ranged between 15.4 and 107.0 ng mL(-1) and 3.9 and 20.4 ng mL(-1) for flavonoids and isoflavones, respectively. Intra-day and inter-day precision (C.V., %) was less than 3.9% and 3.8%, respectively, and accuracy was between 0.03% and 5.0%. Recovery was 70.35-96.58%. The method is very rapid, simple, and reliable, and suitable for pharmacokinetic analysis. It can be routinely used for simultaneous determination of these five isoflavones and seven flavonoids in human urine. The method can also be applied to studies after administration of pharmaceutical preparations containing isoflavones and flavonoids to humans.
建立并验证了一种快速、超高液相色谱（UHPLC）法，用于同时鉴定和分析人尿中的异黄酮染料木黄酮、大豆苷元、大豆苷、葛根素和芒柄花素，以及黄酮类化合物（±）-儿茶素、（-）-表儿茶素、芦丁、橙皮苷、新橙皮苷、槲皮苷和橙皮素。尿样用β-葡萄糖醛酸酶/硫酸酯酶孵育。UHPLC 采用 Hypersil Gold（50×2.1mm，1.9μm）分析柱进行。洗脱液由含 0.05%三氟乙酸的水溶液和乙腈梯度组成。UV 检测在 254nm 和 280nm 处进行。校准曲线在各分析物感兴趣的范围内呈良好线性（r²≥0.9992）。LOD 范围为 15.4-107.0ng/mL 和 3.9-20.4ng/mL 分别用于黄酮类化合物和异黄酮。日内和日间精密度（C.V.%）分别小于 3.9%和 3.8%，准确度在 0.03%-5.0%之间。回收率为 70.35%-96.58%。该方法快速、简单、可靠，适用于药代动力学分析。可常规用于同时测定人尿中的这五种异黄酮和七种黄酮类化合物。该方法也可应用于含有异黄酮和黄酮类化合物的药物制剂在人体给药后的研究。
