Analysis of isoflavones and flavonoids in human urine by UHPLC

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.