Ultimate xb c18 column

Mt was activated by 5% H₂SO₄ for 0.5 hours at 70°C.24 After the exchange of interlayer metal ions with H⁺, Mt was ultrasonically treated for 5 minutes to obtain ultrafine montmorillonite treated with acid (acid-Mt) particles. Then, the acid-Mt was centrifuged and washed with deionized water until the pH was equal to 7.0. The solids were dried at 100°C and crushed.
One gram acid-Mt and 3 g BH were added to 1,000 mL deionized water, and the adsorption process was carried out in a water bath (50°C) for 6 hours. After washing, centrifuging, and drying, solid products of Mt-BH were obtained. DL% (Q) was determined with HPLC (Agilent 1200; Agilent Technologies, Santa Clara, CA, USA) using Equation 1:
$Q=\frac{(C_0-C)\times V_{\text{BH}}}{M_{\text{acid-Mt}}}\times 100\%$ where C₀ and C (mg·mL⁻¹) are the BH concentrations before and after DL into acid-Mt, respectively, M_acid-Mt (mg) is the mass of acid-Mt, and V_BH (mL) is the volume of the BH solution.
The HPLC conditions were as follows: the BH concentration was determined by HPLC. Ultimate^® XB-C18 column (Welch, Austin, TX, USA; 4.60×250 mm, 5 μm) was used. The mobile phase was acetonitrile/trimethylamine (30/70, v/v) with pH 3.0. The detector wavelength, flow rate, column temperature, and injection volume were 275 nm, 1 mL·min⁻¹, 25°C, and 20 μL, respectively.

After MSCs were thawed in a 37°C water bath, 20 µL of sample was pipetted by automatic sampler and analyzed with high-performance liquid chromatography (HPLC). The compounds were separated on an analytical Welch Ultimate XB–C18 column (4.6×250 mm i.d., 5 µm; Welch, Shanghai, China) using a binary gradient delivered by a LC-20AT low-pressure gradient HPLC pump (SHIMADZU, Japan) with acetonitrile as solvent A and 0.1% v/v formic acid water solution as solvent B for the detection of quercetin and its catabolites: 0.01 min, 5% A; 10 min, 10% A; 50 min, 50% A; 65 min, 90% A; 70 min, 90% A; 73 min, 5% A. The flow rate was 0.6 mL/min. Data acquisition was carried out with the software Classic-VP Chromatography Workstation (SHIMADZU, Japan). A diode array detection SPD-M20A plus was applied over the wavelength range of 220–600 nm for peak detection. Compounds were identified by comparison of the UV spectra and the retention times. The following wavelengths were monitored for quantitative analysis: 360 nm for quercetin, 280 nm for metabolites (12 (link), 13 (link)).
The fermentation products separated by the HPLC were used for the further experiment. Those components at new peaks were collected and analyzed by mass spectrometry (MS) (Waters, Milford, MA, USA), and the chromatographic scans of selected multiple reaction monitoring ions (MRM) were used for further identification.

TCDCA conversion and TUDCA yield were detected using HPLC-ELSD. The HPLC analysis was performed with a Waters Acquity SQD (Santa Clara, CA, USA) equipped with a 150 × 2.1 mm, 3 μm Welch Ultimate XB-C18 column. The injection volume was 5 μL. Two solvents (A: 100% methanol; B: 50 mM aqueous ammonium acetate solution) were used at a constant flow rate of 0.8 mL min⁻¹. Elution was performed using a linear gradient from 60 to 90% A for 15 min, from 90 to 60% A immediately, and then held constant for 10 min. The ELSD detection temperature was 80 °C. The detection was carried out in nitrogen environment and the speed of nitrogen was 3.0 L min⁻¹.²¹

Entrapment efficiency and drug-loading rate The entrapment efficiency (EE%) and drug-loading rate (DL%) of BH-Mt/CS NPs were determined by dynamic dialysis technique. In brief, 2 mL of BH-Mt/CS NPs dispersions (2.8 mg·mL⁻¹) were loaded in a dialysis bag with a molecular weight of 12–14 kDa and then immersed in 35 mL normal saline (NS) with a stirring of 120 rpm·min⁻¹ at 34°C. At 25 min (free drug molecules were dialyzed completely), 5 mL of release medium was withdrawn, and the BH concentration was determined by HPLC (Agilent 1200, Agilent Technologies, Santa Clara, CA, USA). Ultimate^® XB-C18 column (Welch, 4.60×250 mm², 5 µm) was used, and the mobile phase was acetonitrile/trimethylamine (30/70, v/v, pH 3.0). The detector wavelength was 275 nm. The flow rate, column temperature, and injection volume were 1 mL·min⁻¹, 25°C, and 20 µL, respectively. The HPLC method was validated with respect to linearity, repeatability, and the limit of quantitation and limit of detection. The EE% and DL% of BH-Mt/CS NPs were calculated according to the Equations (1) and (2), respectively:
$\begin{array}{l}\text{Encapsulation efficiency}\left(\%\right)\\ =\frac{\text{Total amount of BH}-\text{Free BH}}{\text{Total amount of BH}}\times 100\end{array}$
$\begin{array}{l}\text{Drug loading capacity}\left(DL\%\right)\\ =\frac{\text{Total amount of BH}}{\text{Total}\text{weight}\text{of}\text{NPs}}\times 100\%\end{array}$