Mightysil rp 18

XO activity was estimated using caffeine as a probe compound, which was metabolized to 1-methylxanthine (1-MX) and then to 1-methyl uric acid (1-MU) by XO. The molar ratio of urinary excretion of 1-MU acid to 1-MX reflects in vivo intrinsic XO activity (caffeine test) [17 (link)]. First, the rats were administered febuxostat (10 mg/kg) or 1% sodium carboxymethyl cellulose solution, followed by caffeine (30 mg/kg). Urine was collected for 24 h. To determine the concentrations of 1-MU and 1-MX, urine was pretreated as described by Grant et al. [18 (link)]. Water (0.3 mL), 0.4 mg acetaminophen (internal standard), and 0.6 g of ammonium sulfate were added to the urine sample (0.5 mL). The mixture was briefly vortexed. Dichloromethane/isopropanol (5 mL; 85:15, v/v) was added to the mixture and then vigorously vortexed for 30 s. Subsequently, the mixture was centrifuged at 900 x g for 10 min. The organic phase was then removed and evaporated. The residue (20 μL) was resuspended in the HPLC mobile phase and subjected to HPLC on a Mightysil RP-18 (4.6 mm × 150 mm) column (Kanto Chemical Co., Inc., Tokyo, Japan) with ultraviolet detection at 280 nm. The mobile phase consisted of a mixture of 0.5% acetic acid/methanol (92:8, v/v), with a flow rate of 0.5 mL/min. The elution times of 1-MU, 1-MX, and acetaminophen (internal standard) were 15.0, 19.6, and 23.0 min, respectively.

Test compound (2 mg) was suspended with 1 mL of normal saline, pure water, or 5% glucose aqueous solution in glass tube, and the suspension was shaken at 150 rpm for 48 h at 25 °C. An aliquot was filtered by PVDF filter unit (Mini-Uni Prep^TM, GE Healthcare), and the filtrate was diluted with pure water. The diluted sample solution was injected into HPLC system (column (Mightysil RP-18, Kanto Chemical Co. Inc.), UV/vis detector (UV-2077, JASCO), pump (PU-2089, JASCO), and column oven (CO-965, JASCO)) and the peak area was recorded at 254 nm. The concentration of solution was calculated with a calibration curve. Examinations of all conditions were performed three or four times.

Metabolite analyses were performed as described by Sugimoto et al. (2014) (link). Briefly, leaf metabolites were extracted by adding 3 × volume of MeOH containing 1 μg mL^–1 formononetin to ca. 100 mg of ground frozen leaf powder. The extract was injected into the LC-MS (3200 QTRAP, SCIEX, Framingham, MA, United States) or LC-high resolution-mass spectrometry (LC-HRMS; Finnigan LTQ-FT, Thermo Fisher Scientific, Waltham, MA, United States). Chromatographic separation was performed using 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The gradient program was started with 20% B for the initial 10 min, 20 to 50% for the next 10 min, followed by an increase from 50 to 95% in 20 min, and a constant 95% B for the final 5 min, with a constant flow rate of 0.2 mL min^–1 and with an ODS column (Mightysil RP-18, 5 μm, 2 mm × 150 mm, Kanto Chemical, Tokyo, Japan) for LC-MS analysis. The gradient program for LC-HRMS analysis was started with 10% B, followed by an increase to 50% in 50 min, then 90% in 10 min followed by a constant 90% B for the final 5 min with a constant flow rate at 0.5 mL min^–1, and the separation was achieved with an ODS column (TSK-gel ODS-100V, 5 μm, 4.6 mm × 250 mm, TOSOH, Japan).