8510e dth

High-performance liquid chromatography (HPLC) analysis for the simultaneous determination of four reference components in the CRE sample was conducted by using an LC-20A Prominence HPLC system (Shimadzu Co., Kyoto, Japan) equipped with binary pumps, a column oven, an autosampler, and a photodiode array (PDA) detector. In brief, 25 mg of the lyophilized CRE was dissolved in 25 mL of 70% methanol and extracted for 60 min using an ultrasonicator (Branson 8510E-DTH, Danbury, CT, USA), and the extracted solution was then filtered through a 0.2 mm membrane filter (PALL Life Sciences, Ann Arbor, MI, USA). Then, the CRE sample and the four reference components (jatrorrhizine, coptisine, palmatine, and berberine) were subjected to analysis in an HPLC-PDA system. They were separated on a SunFire C18 column (4.6 × 250 mm, 5 mm; Milford, MA, USA) maintained at 30 °C. The mobile phase was eluted with 30 mM ammonium bicarbonate and 0.1% (v/v) aqueous triethylamine (A) and acetonitrile (B) in gradient elution mode. The flow rate was 1.0 mL/min with the following linear gradient: 0 to 15 min, 90–75% A and 10–25% B; 15 to 25 min, 75–70% A and 25–30% B; 25 to 40 min, 70–55% A and 30–45% B; 40 to 45 min, 55% A and 45% B; and 45 to 60 min, 55–90% A and 45–10% B. CRE was detected using a photodiode array at 200–500 nm (Figure 1).

Simultaneous determination of the 11 marker components for THD quality control was performed using the LC-20A Prominence HPLC system (Shimadzu Co., Kyoto, Japan) coupled with a photo-diode array (PDA) detector and Lab Solution software (version 5.53, SP3, Kyoto, Japan). Analyte separation was carried out using the Sun Fire C18 analytical column (4.6 × 250 mm, 5 μm; Milford, MA, USA), constantly maintained at 40 °C. The mobile phase for the efficient separation and analysis of the marker components in the THD sample consisted of distilled water (A) and acetonitrile (B), both containing 0.1% (v/v) formic acid. The gradient eluting conditions were as follows: 0–30 min, 5–60% B; 30–40 min, 60–100% B; 40–45 min, 100% B; 45–50 min, 100–5% B. For the analysis, 200 mg of the lyophilized THD sample was dissolved in 20 mL of distilled water, and the solution was extracted using an ultrasonicator (Branson 8510E-DTH, Denbury, CT, USA) for 60 min. The extracted solution was filtered using a 0.2-μm membrane filter (PALL Life Sciences, Ann Arbor, MI, USA) before sample injection for HPLC analysis.