Tc c18 2

The separation and quantification of the four purines and uric acid were performed using an Agilent 1260II ultra-HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a G7104C quaternary pump, G7129C autosampler, G7116A column heater, and variable wavelength detector (VWD). An Agilent TC-C18 (2) (4.6 mm × 250 mm, 5.0 µm) was used as the analytical column, and the injection volume was 10 µL. In order to establish a suitable HPLC detection method, the following parameters: mobile phase (7 × 10⁻³ M KH₂PO₄, 0.02 M KH₂PO₄, and methanol-water solutions); flow rate (0.8, 1.0 and 1.2 mL/min); column temperature (25 °C, 28 °C, 30 °C, and 35 °C) and absorption wavelength have been optimized. Data were collected and processed using OpenLab CDS Data Analysis software (product version 2.5; Agilent Technologies, Santa Clara, CA, USA).

Silymarin content was determined using HPLC. Briefly, 100 mg of milk thistle oil is taken and adsorbed for 2 min on an activated C18 SPE column (1000 mg, 6 mL), after which 10 mL of n-hexane is added for elution. The receiving solution is then discarded, and the process is repeated with 10 mL of ethanol. At 45 °C, the ethanol receiver liquid is spun dry in an evaporator. It is then dissolved in a 2 mL solution of methanol and detected using HPLC. The chromatographic detection conditions are as follows [38 (link)]: chromatographic column—Agilent TC-C18(2), 250 × 4.6 mm, 5 μm; mobile phase A—methanol (chromatographic grade) and mobile phase B—methanol (20% ultrapure water); injection volume 20 μL; column temperature 45 °C; flow rate, 0.6 mL/min; and detection wavelength, 288 nm. The elution gradient was 0–8 min, 80% B; 8–10 min, 10% B. In addition, silibinin was used as the standard to draw a standard curve, and the regression equation was Y = 7.33472 × 10⁷ X − 514722, R² = 0.9998.