300sb c18 column

Samples were simultaneously denatured and reduced using guanidine hydrochloride and dithiothreitol (6 M and 10 mM final concentration, resp.; 30 min at room temperature (RT)). Next, samples were alkylated using 4-vinylpyridine (3 mM final concentration; 90 min at RT, protected from light). Chromatographic separation was performed using a Zorbax 300SB-C18 column (4.6 mm × 100 mm; 3.5 μm) installed on an Agilent 1100/1200 HPLC system in gradient mode. The starting mobile phase was 95%  A and 5%  B, where “A” is 0.1% (v/v) trifluoroacetic acid (TFA) in water and “B” is 0.1%  TFA in 25% isopropyl alcohol, 75% acetonitrile. A nonlinear gradient from 5%  B to 52%  B was performed over 67 minutes, followed by a wash step with 100%  B. The flow rate was 1 mL min⁻¹, and the column was maintained at 30°C. A 25 μg protein load was used.

The in vitro drug release behaviors of the DNVs were analyzed using the modified dialysis method shown in Figure 1, as previously described [25 (link)]. Briefly, 1 mL IPC-DNVs or IPC-DNV gel (pre-melted at 37 °C) was transferred to a dialysis bag (diameter: 16 mm, molecular weight cut-off: 100 KD; Spectrum Chemical, New Brunswick, NJ, USA), and the bag was gently pressed to expel air and closed tightly at both ends to maintain a consistent surface area for each bag. The dialysis bag was placed in a centrifuge tube with 15 mL PBS (pH 7.4) and shaken in a constant temperature shaker at 37 °C and 80 rpm. At 1, 3, 8, and 12 h, 15 mL aliquots of release medium were removed and replaced with 15 mL of fresh, preheated medium. Each experiment was conducted in triplicate. The concentration of insulin was determined using reverse-phase, high-performance liquid chromatography with an Agilent 1200 series HPLC system (Santa Clara, CA, USA) and a 300SB-C18 column (4.6 mm × 250 mm, 5 μm.).

Toxin depletion experiments depicted in Figures 2C, 3F, and 5A were performed as reported previously (18 (link)). Specifically, uninjected defolliculated stage V or VI oocytes (100 nos.) were incubated with 4 nmol of toxin dissolved in a 400 μl solution of HEPES (20 mM) buffer at pH 7.4 containing NaCl (50 mM), KCl (50 mM), MgCl₂ (1 mM), and BaCl₂ (0.3 mM) for 1 h at room temperature. Control experiments involved incubating the toxin under identical conditions without oocytes. Subsequently, the supernatant (200 μl) was removed and spiked with a solution of 2-nitrophenol (2.5 μl of a 2.5 mg/ml solution in water) used as an internal standard. A 100 μl aliquot of this mixture was injected into the reverse phase HPLC column (Agilent 300SB-C18 column, 300 Å pore size) and eluted using a water-acetonitrile (0.1% TFA) linear gradient (5–65% acetonitrile over 30 min). This experimental protocol was performed three times and the fractional depletion was calculated as follows: $\text{Fraction}\text{depletion}=\left({\mathrm{R}}_{\text{control}}{-\mathrm{R}}_{\text{oocytes}}\right)/{\mathrm{R}}_{\text{control}}$
R_control is the averaged ratio of the area under the peak for the toxin and the area under the peak for 2-nitrophenol in the control experiments, and R_oocytes is the ratio of the same parameters obtained from the oocyte samples (n = 3).

A Waters ACQUITY UPLC system was coupled with a Thermo Fisher UltiMate 3000 UHPLC and an Aglient ZORBAX 300SB-C18 column (250 × 4.6 mm, 2.6 μm) and operated at a flow rate of 0.2 mL·min⁻¹ for quantitative analysis. The mobile phase consisted of buffer A (0.1% FA in H₂O) and buffer B (0.1% FA in acetonitrile), and the elution was performed with a mixture of buffer A and B in a ratio of 85:15 (v/v). The ESI voltage was 4.0 kV, the capillary temperature was 320°C, and SRM ion transition was selected based on an m/z value of 103.2.

The concentration of TC in the supernatant filtrate was analyzed directly using an Agilent™ 1260 Series high performance liquid chromatography (HPLC) system (Agilent, Santa Clara, CA, USA) equipped with DAD operated at wavelength of 280 nm and an ZORBAX 300SB-C18 column (4.6 × 150 mm, 5 μm). Mobile phase was composed of 0.01 M oxalic acid (pH 4.0), methanol and acetonitrile (66.6:20:13.4, v/v/v) at a flow rate of 0.3 mL min⁻¹. The column oven temperature was set at 30 °C, and the injection volume was 20 μL. The calibration curve was significantly linear in the concentration range of 2-100 µM (p < 0.05, r² = 0.9993).

RP-UPLC was used to separate HNW-GS and LMW-GS based on the recent reports [56 (link),69 (link)]. The samples were performed on an Agilent 1100 using a Zorbax 300SB-C18 column (300 A° pore size and 5 mm particle size).