Kromasil 100 5 c18 column

The quantification of five phthalides of VOC in formulation samples was determined by a validated reversed-phase HPLC-UV method (Shimdzu, Japan). The HPLC system consisted of a LC-20AT pump, an SPD-M20A PAD-visible detector, a SIL-20A injector with a 20 μL loop and an LC solution workstation. The mobile phase consisted of a mixture of ultrapure water (A) and acetonitrile (B) (0~10 min, 38~42% B; 10~36 min, 42~45% B; 36~55 min, 45~48% B; 55~60 min, 48~38% B) was delivered with a flow rate of 1 mL/min, and separation was performed using an Akzo Nobel Kromasil 100-5-C18 column (4.6 mm × 250 mm, 5 µm) with a sample injection volume of 20 µL. The analysis was performed at 30 °C with UV detection at 280 nm for senkyunolide A(SA) and Z-ligustilide (ZL), and 230 nm for neocnnolide (NOL), n-butylphthalide (NBP) and butenylphthalide (BP) [22 (link)].

5-n-Nonadecylresorcinol, 5-n-heneicosylresorcinol, pinellic acid and tachioside from the water extract of WB were isolated and identified as described previously [21 (link)]. Conditions for High performance liquid chromatography (HPLC) were described as follows: Waters Alliance e2695 separating module (Waters Co., Milford, MA, USA) using photodiode array detector (Waters 2998) with autosampler and column oven was used for the analysis. Separation was achieved using a Kromasil 100-5-C18 column (5 µm, 250 × 4.6 mm i.d., AkzoNobel, Bohus, Sweden). The mobile phase consisted of water-trifluoroacetic acid (99.95:0.05; v/v) (solvent A) and acetonitrile (solvent B). The elution was performed using the following gradient: initial 95:5 (A:B v/v); 10 min 95:5 (A:B v/v); 30 min 50:50 (A:B v/v); 45 min 0:100 (A:B v/v); and 70 min 0:100 (A:B v/v). The mobile phase flow rate was 1.0 mL/min. The injection volume was 10 μL, and the column temperature was set at 30 °C. All operations, including data acquisition and analysis, were controlled by Empower™ 3 chromatography data software (Waters Co., Milford, MA, USA). The HPLC chromatogram of the water extract of WB was presented in Figure 4a.

Waters Alliance e2695 separating module (Waters, MA, USA) using photodiode array detector (Waters 2998) with autosampler and column oven was used for the analysis. Separation was achieved using a Kromasil 100-5-C18 column (5 μm, 250 × 4.6 mm i.d., AkzoNobel, Bohus, Sweden). The mobile phase consisted of water–trifluoroacetic acid (TFA, 99.95:0.05; v/v) (solvent A) and acetonitrile (solvent B). The elution was performed using the following gradient: initial 90:10 (A:B v/v); 40 min 50:50 (A:B v/v); and 60 min 0:100 (A:B v/v). The mobile phase flow rate was 1.0 mL/min. The injection volume was 10 μL, and the column temperature was set at 30 °C. All operations, including data acquisition and analysis, were controlled by Empower™ 3 chromatography data software (Waters Co., MA, USA).

The polysaccharide sample DOPs were dissolved with distilled water to the concentration of 1 mg/mL. And the pH of the samples was adjusted to 7.0 using NaOH solution after the 3 M HCl was added to the sample solution, while the concentration of mixed monosaccharide standards, galactose (Gal), mannose (Man), glucose (Glu), and rhamnose monohydrate (Rha), was adjusted to 0.45 mg/mL. The solutions of samples and mixed monosaccharide standards were converted to their derivatives with 0.5 M PMP. All the solutions were allowed to react at 70°C in an oven for 100 min and then cooled to ambient temperature (25°C) and added with 0.3 M HCl. Last, chloroform was added and mixed via vortexing, followed by centrifugation at 3000 rpm for 10 min to discard chloroform. This procedure was repeated 3 times until no colour was evident in the chloroform layer. The lower water-phase layers were the sample solution and mixed monosaccharide standard solution.
The monosaccharide compositions of DOPs were analysed by high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) equipped with a Kromasil 100-5-C18 column (4.6 × 250 mm, 5 μm) (Akzo Nobel, Sweden). The mobile phase was composed of acetonitrile (solvent A) and 0.1 M phosphate buffer (solvent B) at a ratio of 15.5 : 84.5 under isocratic elution, and the flow rate was 0.8 mL/min.