Cosmosil 5c18 ms 2 reversed phase column

Quantification of the possible active constituents in ARE, aloe-emodin, chrysophanol, emodin, physcion, rhein, and psoralen, was provided by Herbiotek Co., Ltd., New Taipei City, Taiwan. Twenty milliliter of ARE was filtered through 0.22-μm membranes before analyzed. The Waters HPLC system (Milford, Massachusetts, USA) included Waters 600 pump system, Waters 2996 photodiode array detector, Waters 717 plus autosampler, and Sugai U-620 column oven (Wakayama City, Japan). Cosmosil 5C18-MS-II reversed phase column (5 μm, 4.6 mm × 250 mm, Nacalai Tesque, Japan) equipped with LiChrospher RP-18 end-capped guard column (5 μm, 4.0 mm × 10 mm, Merck, Germany) was used as the stationary phase. The gradient elution was consisted of 10 mM phosphate buffer, acetonitrile, and water. The flow rate was 1 mL/min, and the column temperature was maintained at 35 °C. UV 246 nm was used for detection of psoralen (a furanocoumarin isomer), with a retention time of 14.5 min. UV 270 nm was used for detection of aloe-emodin, rhein, emodin, chrysophanol, and physcion, with a retention time of 89.1, 94.1, 107.4, 114.9, and 118.1 min, respectively. Data are presented as the concentration (μg/mL) of each constituent in ARE.

The Waters HPLC system (Milford, Massachusetts, United States) was comprised of Waters 600 pump system, Waters 2996 Photodiode array detector, Waters 717 plus Autosampler, and Sugai U-620 Column oven (Wakayama City, Japan). Cosmosil 5C18-MS-II reversed phase column (5 μm, 4.6 × 250 mm, Nacalai tesque, Japan) equipped with Lichrospher RP-18 end-capped guard column (5 μm, 4.0 × 10 mm, Merck, Germany) was used as the stationary phase. The gradient elution was composed of eluents A, B, and C (A: H₂O/KH₂PO₄/10% H₃PO₄ = 1000 ml/2.72 g/1 ml; B: Acetonitrile; C: H₂O) according to the following profile: 0–30 min, 90%–75% A and 10%–25% B; 30–40 min, 75%–65% A and 25%–35% B; 40–55 min, 65%–0% A, 35%–75% B and 0%–25% C; 55–60 min, 75%–10% B and 25%–90% C; 60–65 min, 0%–90% A, 10% B and 90%–0% C. The gradient elution was used for 3D fingerprint analysis and quantification of puerarin (250 nm), daidzin (250 nm), daidzein (250 nm), chlorogenic acid (320 nm) and 3,5-dicaffeoylquinic acid (325 nm). The flow rate was 1 ml/min, and the column temperature was maintained at 35°C.