Lc 20at

The main polyphenols in ROE were determined using high-pressure liquid chromatography (HPLC, SHIMAZU LC-20AT liquid chromatography, Japan) with a photodiode array (PDA, SHIMAZU SPD-M20A) detector. Separation and identification were based on our previous report^{16 (link)} and performed with a COSMOSIL 5C18-MS-II (4.6 mm × 250 mm, 5 μm) at 30 °C with a gradient elution solution A, composed of a formic acid–water solution (0.1% formic acid), and solution B, comprising an acetonitrile and formic acid–water solution (0.1% formic acid) (6 : 4; v/v), which were delivered at a flow rate of 0.7 mL min⁻¹ as follows: 0 min, 96% (A); 180 min, 70% (A); 205 min, 96% (A); and 210 min, 0% (A). The UV spectra of every identified peak were recorded at 280 nm. The tested standards, including procyanidin B3 (1), EGC (2), catechin (3), epicatechin (4), (−)-fisetinidol-(4α,8)-(−)-catechin (5), (4α,8)-(−)-fisetinidol-(−)-epicatechins (6), and (+)-guibourtinidol-(4β,8)-epicatechin (7), were made by our laboratory. These compounds were isolated and identified from ROE by our group and the purity of each standard was higher than 95%.

Twenty tablets were taken at random from each lot and weighed before being placed in a mortar and being porphyrized with a pestle. A quantity equivalent to 5 mg of IMM-H014 (40 mg of powder) was pulled out with 100 mL of hydrochloric acid solution (0.1 N) and supersonically extracted over 30 min. Then, the resulting 10 mL solution was filtered through a polytetrafluoroethylene filter membrane (PTFE, 0.45 μm pore size, Jinteng, Tianjin, China). A C18 column was used in the stationary phase. The collected samples (20 μL) were analyzed using HPLC (Shimazu LC-20AT, Kyoto, Japan) with a UV detector wavelength of 230 nm. IMM–H014 was separated under a mobile phase consisting of ammonium formate PBS (pH 4.0) to acetonitrile at a 60:40 (v/v) ratio using a C18 column (4.6 mm × 25 cm, 5.0μm). The flow velocity was 0.7 mL/min and the column temperature was 35 °C. The linearity of IMM-H014 ranged from 20.01 μg/mL to 60.02 μg/mL. The validation was conducted following the ICH guidelines.

The HPLC analysis was performed on a Shimadzu-LC-20AT (Japan) with an Ultimate® XB-C18 column (4.6 mm × 250 mm I.D., 5 μm, Welch Materials, Inc., China) at a flow rate of 0.8 mL/min, using the mobile phase of (A) 0.1% formic acid in deionized H₂O and (B) 100% MeCN. The gradient settings for separating the products and substrates were 0–20 min 10% B to 50% B, 20–25 min 50% B to 100% B, 25–28 min 100% B to 10% B, and 28–35 min 10% B to 10% B. The products were further confirmed using an LTQ XL Orbitrap mass spectrometer (Thermo Fisher Scientific Inc.) The MS/MS analysis was carried out in a positive ionization mode with 35% relative collision energy. The relative activities of the mutants were determined by HPLC and calculated by the product’s peak area dividing the wild-type’s peak area. All experiments were performed in triplicate.

The extraction process of TMZY extracts was conducted as previously reported. Briefly, one dose of TMZY medicines were added in 10 volumes of water, soaked for 60 min, and decocted two times (3 h at the first time and 2 h at the second time). The two decoctions were then combined together. In order to control the quality of extracts, the fingerprint spectrum was measured by a high-performance liquid chromatography (HPLC) method, and the chromatographic profile of the TMZY extracts is presented in Figure 1. The analyses were conducted by Shimadzu system (LC-20AT, Shimadzu Corp., Japan) equipped with an auto-sampler, a quaternary gradient pump, and a SPD-20A detector. The mobile phase was acetonitrile (A) – 0.2% formic acid solution (B). The mobile phase gradient elution program is shown in Table 1 and the flow rate was 1.0 ml/min. The chromatographic column was Shim-pack VP-ODS C18 (4.6 mm × 250 mm, 5 μm) with working temperature of 30°C. The detector wavelength was set at 254 nm. The peak assignment in Figure 1 is listed in Table S1.

The HPLC equipment used for the quantitative analysis of the two flavones, kuwanon G and morusin, in M. alba L. was a Shimadzu LC-20A series (Kyoto, Japan) consisting of a pump (LC-20AT), degassing unit (DGU-20A3R), column oven (CTO-20A), auto sampler (SIL-20A), and PDA detector (SPD-M20A). The acquisition and processing of chromatographic data was performed using the LabSolution software (Version 5.53, SP3, Shimadzu, Kyoto, Japan). The column used for the separation of kuwanon G and morusin was the Phenomenex Gemini C18 analytical column (250 mm × 4.6 mm, 5 μm, Torrance, CA, USA), which was maintained at 45 °C. The mobile phase for the efficient separation of analytes was composed of 0.1% (v/v) aqueous formic acid (A) and acetonitrile (B), and flowed from the initial 20% B to 90% B for 50 min. The flow rate was kept constant at 1.0 mL/min, the injection volume was 10 μL, and the PDA scanned for quantification was 190–400 nm. For HPLC analysis of the two biomarker compounds in M. alba L., 500.0 mg of the ground raw M. alba L. sample material was dissolved in 20 mL of 70% methanol and extracted for 60 min at 25°C using a Branson ultra-sonicator, 8510E-DTH (Danbury, CT, USA). Then, the extracted solution was filtered through a 0.2 μm membrane filter (Pall Life Sciences, MI, USA), before injection into the HPLC equipment.

The TEM and SEM images of the MGAC were examined on an transmission electron microscope (JEM-2100, JEOL, Japan) with a high voltage of 200 kV and an scanning electron microscopy (JSM-6360LV, JEOL, Japan) with 5.0 kV, respectively. The specific surface area was calculated by the Brunauer–Emmett–Teller (BET) method and the pore volume and pore size distribution were estimated based on Barrett–Joyner–Halenda (BJH) model. The X-ray photoelectron spectroscopy (XPS) spectrum of this adsorbent was obtained by an XPS instrument (ESCALAB 250Xi, Thermo Scientific, USA) with the Al-KR as the excitation source. The zeta potentials of the MGAC were determined by a zeta potential instrument (Nano ZS, Malvern, UK) following the method of Wang et al.^{25 (link)} Fourier-transform infrared (FTIR) spectra of the MGAC before and after adsorption were recorded from samples in the wavenumber range of 4000–400 cm⁻¹ in KBr pellets on a FTIR spectrometer (NICOLET 6700, Thermofisher, USA). The concentrations of TBBPA in solutions were determined by a high performance liquid chromatography (HPLC) (LC-20AT, Shimadzu, Japan) equipped with an UV detector and a C18 reverse-phase column (Inertsil ODS) (250 mm × 4.6 mm i.d., particle size 5 μm) using methanol/water (80 : 20 (v/v)) mobile phase. The pH values of solutions were measured with a pH meter (HQ30d, HACH, USA) after calibration.