Prominence lc 20a series hplc system

HPLC analysis of the seven characteristic constituents in MHT was performed using a Shimadzu Prominence LC-20A series HPLC system (Kyoto, Japan) coupled with a photodiode array detector and LabSolutions software (Version 5.54 SP3, Shimadzu, Kyoto, Japan). A Waters Sun Fire C₁₈ analytical column (250 × 4.6 mm, 5 μm; Milford, MA, USA) as the stationary phase, maintained at 40°C, was used for the separation of the main components. The mobile phases consisted of 0.1% (v/v) trifluoroacetic acid in distilled water (A) and acetonitrile (B). The elution gradient used for chromatographic separation was as follows: 10%–60% B for 30 min, 60%–100% B for 30–40 min, 100% B for 40–45 min, 100%–10% B for 45–50 min, and 10% B for 50–60 min. The flow rate was set to 1.0 mL/min, and the injection volume was 10 μL.

The quantitative determination was performed with a Shimadzu Prominence LC-20A series HPLC system (Kyoto, Japan) consisting of a solvent delivery unit (LC-20AT), on-line degasser (DGU-20A₃), column oven (CTO-20A), auto sample injector (SIL-20AC), and photodiode array detector (SPD-M20A). Data were collected and processed using LCsolution software (version 1.24; Shimadzu, Kyoto, Japan). The marker compounds were separated on a Gemini C₁₈ column (250 mm × 4.6 mm; particle size 5 μm; Phenomenex, Torrance, CA, USA) maintained at 40°C. The mobile phases consisted of 0.1% (v/v) trifluoroacetic acid in distilled water (A) and acetonitrile (B). The gradient flow was as follows: 10–60% B for 0–30 min, 60–100% B for 30–40 min, 100% B for 40–45 min, and 100–10% B for 45–50 min. The analysis was conducted at a flow-rate of 1.0 mL/min with PDA detection at 254 nm (glycyrrhizin), 275 nm (liquiritin, baicalin, wogonoside, baicalein, and wogonin), and 350 nm (coptisine, palmatine, and berberine). The injection volume was 10 μL.

The lyophilized fractions of the CWE-EA, HWE-EA, and ME-EA were reconstituted in 1 mL of 20% aqueous methanol (v/v) and passed through a 0.2 μm nylon filter. Eleven standards were included for flavonoids (quercetin and kaempferol), triterpenoids (arjunic acid and euscaphic acid), polyphenols (caffeic acid, chlorogenic acid, ferulic acid, gallic acid, tannic acid, and coumarin), and kojic acid. Chromatographic separations were performed on an Agilent Zorbax Eclipse XDB-C18 (Agilent, Santa Clara, CA, USA) column (4.6 mm × 150 mm, 5 μm). Samples (10 μL) were injected into the HPLC instrument (Shimadzu Prominence LC 20A series HPLC system, Shimadzu Corp, Kyoto, Japan) with a PDA detector. The mobile phase for CWE-EA, HWE-EA, and ME-EA consisted of 0.1% phosphoric acid in water (solvent A) and 0.1% phosphoric acid in acetonitrile (solvent B). Elution from the column was achieved with the following gradient: 0–5 min, 97% A and 3% B; 15–20 min, 90% A and 10% B; 30–40 min, 50% A and 50% B; 40.1–50 min, 97% A and 3% B. The preparative system was run for 40 min of the total running time at a constant flow rate of 0.8 mL/min at ambient temperature, and the spectrum was monitored at 272 nm. The identification of each compound was based on a combination of the retention time and UV spectral matching.

The lotion sample was analyzed using a Shimadzu Prominence LC-20A series HPLC system (Shimadzu Co., Kyoto, Japan), comprising an LC-20AT pump, CTO-20A column oven, SIL-20AC autosampler, and SPD-M20A PDA detector. The acquired chromatographic data were converted and processed by LC solution software (Version 1.24, SP1; Shimadzu, Kyoto, Japan). The column used for the separation of the analytes was an XBridge C18 3.5 um, 4.6 × 100 mm HPLC column (Waters Corp., Milford, MA, USA), maintained at 40 °C. The mobile phase consisted of the MeOH: 50 mM phosphate buffer (pH 2.5) at a ratio of 4:96 with a flow rate of 0.5 mLmin⁻¹. All analyte absorbances were detected at 280 nm with an injection volume of 20 μL.