Sunfire c18 analytical column

The drug adsorption capability of fresh and decellularized livers was measured by incubating with doxorubicin solutions in 24‐well transwell plates. Specifically, dried powders of liver samples (≈5 mg) placed in the transwell inserts (0.8 µm, Corning Co., Corning, NY, USA) were incubated with 1.5 mL of 250 µg mL⁻¹ doxorubicin solutions at 37 °C for 24 h until the tissues were saturated with doxorubicin. The tissue powders were observed under microscope while the doxorubicin in the compartment was tested using high performance liquid chromatography (HPLC) system (Waters, Milford, MA, USA) equipped with a Waters Sunfire C18 analytical column (5 µm, 4.6 × 150 mm) and a UV/vis detector set at 254 nm. The drug samples were mixed with acetonitrile (1:1 v/v) to precipitate soluble proteins, and then centrifuged at 12 000 rpm for 15 min. The collected supernatant was diluted 500 times by mobile phase for HPLC characterization. The drug adsorption capability of liver tissues was calculated by the decrement of doxorubicin concentration in the compartment.

Chromatographic analysis of the marker components in MHT was performed using the Shimadzu Prominence LC-20A series HPLC system (Kyoto, Japan) equipped with photodiode array (PDA) detector and Lab Solution software (Version 5.54 SP3, Shimadzu, Kyoto, Japan). Waters SunFire C₁₈ analytical column (250 × 4.6 mm; 5 μm, Milford, MA, USA) was used for the separation of the main components as the stationary phase and maintained at 40°C. The mobile phases consisted of 0.1% (v/v) trifluoroacetic acid in distilled water (A) and acetonitrile (B) and the gradient elution for chromatographic separation was as follows: 10–60% B for 0–30 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 1.0 mL/min and injection volume was 10 μL.

For HPLC analysis, we used the HPLC system equipped with a photodiode array (PDA) detector (Waters Alliance e2695, PDA #2998, Waters Corp., Milford, MA, United States). The data were acquired and processed using Empower software (version 3; Waters Corp.). For chromatographic separation of the three compounds, a Sunfire C₁₈ analytical column (250 × 4.6 mm, 5 μm, Waters Corp.) was used, which was maintained at 35°C. The gradient conditions were 10–23% B for 30 min, 23–100% B for 10 min, and 100% B for 10 min. The mobile phases consisted of two solvents: 0.1% (v/v) TFA in water (A) and acetonitrile (B). For scanning chromatograms, PDA detection was performed at 210–400 nm. Column were used at flow rate of 1.0 ml/min and injected volume of 10 μL, respectively.

Quantitative analysis of the GMSYS sample was performed using an LC-20A Prominence HPLC system (Shimadzu Corp., Kyoto, Japan) equipped with a solvent delivery unit, an on-line degasser, a column oven, an autosampler, and a photo diode array (PDA) detector. Data were acquired and processed using LabSolution software (version 5.54, SP3; Shimadzu Corp.). Separation was achieved on a SunFire C₁₈ analytical column (250 × 4.6 mm; particle size 5 μm, Waters, Milford, MA, USA) as the stationary phase at a column temperature set to 40 °C. The mobile phases consisted of 0.1 % (v/v) formic acid in water (A) and 0.1 % (v/v) formic acid in acetonitrile (B). The gradient sequence and elution conditions were as follows: 5–60 % B for 0–30 min, 60–100 % B for 30–40 min, 100 % B for 40–45 min, 100–10 % B for 45–50 min, with a reequilibrium time of 10 min. The flow-rate was 1.0 mL/min, and the sample injection volume was 10 μL. For HPLC analysis, 200 mg of lyophilized GMSYS extract was dissolved in 20 mL of distilled water and then the solution diluted 10-fold for quantitative analysis of geniposide and paeoniflorin. Samples were filtered through a SmartPor GHP 0.2 μm syringe filter (Woongki Science, Seoul, Korea) before application onto the HPLC column.

The rBmpD and LF-rBmpD samples were heated and centrifuged, and the supernatant was directly used for LC-MS analysis with an Agilent 1100 series LC system. The analytical method was modified from the method described by Ren and colleagues (42 (link)). Separations were conducted using gradient elution on a SunFire C₁₈ analytical column (2.1 by 150 mm; particle size, 3.5 μm; Waters, Milford, MA, USA). Mobile phases were 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B). The gradient conditions were 5% solvent B (0 to 12 min), from 5% to 80% solvent B (12 to 13 min), 80% solvent B (13 to 18 min), from 80% to 5% solvent B (18 to 18.5 min), and 5% solvent B (18.5 to 25 min). The flow rate was 0.25 ml/min. Retention times for adenosine and inosine were 4.7 and 7.7 min, respectively (Fig. 4A).
MS detection was performed in selected-ion monitoring mode with a single quadrupole mass spectrometer (HP 1100 LC/MSD). Ionization was based on electrospray ionization in positive-ion mode. The capillary voltage was 4.0 kV, and the drying gas temperature was 350°C. The selected ions for adenosine and inosine were m/z 268.0 and 269.0, respectively (Fig. 4A). These masses correspond to the protonated molecules, [M+H]⁺. Adenosine was also detected as m/z 269.0, due to its isotopic distribution.

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.