Eclipse xdb

HPLC-UV chromatography system (Shimazu LC-20AD, Kyoto, Japan) was employed for determination of Tripeptide-3. A reversed-phase C-18 column, (Eclipse XDB, 4.6 mm × 250 mm, 5 µm, Agilent, CA, USA) with a gradient-elution programmed analysis employed the mobile phase consisted of 0.1% trifluoroacetic acid and acetonitrile (ACN) as polar and non-polar mobile phases, respectively. The step gradient elution at a flow rate of 1 mL/min was performed by increasing the ACN from 8% to 16% for 21 min., then to 50% in 4 min, holding at this composition for 3 min, before decreasing ACN to 8% and holding at this composition, before a next injection that led to a total analytical time of 35 min per sample. The column temperature was maintained at 30 °C. A sample solution of 50 µL was injected into the column. The detection was carried out at 215 nm with a UV detector. The HPLC method validation was performed; the assay was linear (coefficient of determination, R² > 0.999) in the Tripeptide-3 concentration range of 0.312–50.0 µg/mL with the lowest quantitation concentration of 312 ng/mL. An accuracy (% recovery) in the range of 90–106% was obtained with the intra- and inter-day precision (RSD) of less than 2.18%. The stability of the Tripeptide-3 in various pH of the colloidal dispersion and 24 h in autosampler was also determined.

The HPLC system included chromatographic pumps (LC-20AT; Shimadzu Co., Kyoto, Japan), an autosampler (SIL-20AC; Shimadzu Co., Kyoto, Japan) and a UV–Vis detector (SPDM20A; Shimadzu Co., Kyoto, Japan). A Waters Acquity C¹⁸ column (50 × 2.1 mm, particle size 1.7 μm, Eclipse XDB, Agilent, Palo Alto, CA, USA) was used for sample analysis. The mobile phase consisted of potassium dihydrogen phosphate (10 mM, pH = 3) and acetonitrile (55:45, v/v). The flow rate was set to 0.2 mL/min, and the injection volume was 5 μL. The temperature in the autosampler was set to 40 °C. The UV–Vis detector scanned from 190 to 300 nm, and the chromatographic profiles were monitored at 265 nm for regorafenib and diethylstilbestrol (internal standard (IS)).

Commercial reagents and solvents were obtained from Sigma-Aldrich or Fisher Scientific unless otherwise noted. BG-NH₂ and BC-NH₂ were acquired from New England Biolabs (NEB), and HaloTag amine (O2) ligand was purchased from Promega. ATTO 674N NHS ester, Alexa Fluor 594 NHS ester, and Cy2 bis-NHS ester were obtained from Sigma-Aldrich, Life Technologies, and GE Healthcare Life Sciences, respectively. All solvents were purchased in septum-sealed bottles stored under an inert atmosphere. Reactions were monitored by LC/MS (Phenomenex Kinetex 2.1 mm × 30 mm 2.6 μm C18 100 Å column; 5 μL injection; 5–98% MeCN/H₂O, linear gradient, with constant 0.1% v/v formic acid additive; 6 min run; 0.5 mL/min flow; ESI; positive ion mode). Reaction products were purified by preparative reverse phase HPLC (Phenomenex Gemini–NX 30 mm × 150 mm 5 μm C18 column). Analytical HPLC analysis was performed with an Agilent Eclipse XDB 4.6 mm × 150 mm 5 μm C18 column under the indicated conditions.

Waters arc HPLC consisted of quaternary pump and PDA detector model 2998. The stationary phase was Agilent Eclipse XDB (250 mm×4.6 mm, 5 μm) C18 column (United States). pH-meter; Digital pH/MV/TEMP/ATC meter, Model- 5005, Jenco Instruments (California, USA).

The standard ginsenosides, namely, Rg1, Re, Rb1, Rb2, Rc, and Rd, were purchased from Shanghai Tauto Biotech Company (Shanghai, China). The standard solutions were dissolved in methanol (Fair Lawn, NJ, USA) for further experiments. Ten samples of P. ginseng roots (4-year-old) from each plot were pooled and considered one sample. P. ginseng extract preparation was performed according to the description of Dong et al. [22 (link)]. Ginsenoside contents were determined through high-performance liquid chromatography (Agilent 1260, USA) using a system equipped with a binary pump, an online degasser, a column compartment, and an auto plate sampler. The reA C18 reversed phase column (250 mm × 4.6 mm, i.d. 5 µm; Eclipse XDB, Agilent, USA) was used for separation. The column temperature was kept at 25 °C, and the flow rate and wavelength were 1.0 ml min⁻¹ and 203 nm, respectively. The gradient was composed of water and acetonitrile. The linear gradient was set as follows: 0–12 min for 19% acetonitrile and 12–60 min for 19–40% acetonitrile.