Vp ods c18 column

¹H and ¹³C NMR were recorded on a Bruker 400 spectrometer. ¹H NMR data are reported as follows: chemical shift in ppm (δ), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), coupling constant (Hz), relative intensity. ¹³C NMR data are reported as follows: chemical shift in ppm (δ). HPLC-MS analyses were performed on a Shimadzu-2020 LC-MS instrument using the following conditions: Shim-pack VPODS C18 column (reverse phase, 150 × 2.0 mm); 80% acetonitrile and 20% water over 6.0 min; flow rate of 0.4 mL/min; UV photodiode array detection from 200 to 300 nm. The reaction was irradiated at the required ceiling temperature (the reaction temperature was monitored by an external surface sensor using the Biotage Initiator reactor) using maximum power for the stipulated time. Then it was cooled to 50 °C with gas jet cooling. The products were purified by Biotage Isolera™ Spektra Systems and Hexane/EtOAc solvent systems. All reagents and solvents were obtained from commercial sources and used without further purification.

All reagents and solvents were purchased from Sigma-Aldrich (Milwaukee, WI, USA) and Alfa Aesar (Ward Hill, MA, USA). All experiments involving moisture-sensitive compounds were carried out in an argon atmosphere. Analytical thin-layer chromatography was performed on precoated silica gel F₂₅₄ thin layer chromatography (TLC) plates (E, Merck, Kenilworth, NJ, USA) with visualization under UV light. The final products were purified by preparative medium-pressure liquid chromatography (MPLC) (Biotage Isolera One instrument) on a silica column (Biotage SNAP HP-Sil) spectrometer (Biotage, Uppsala, Sweden). Nuclear magnetic resonance (NMR) analyses were performed using a Bruker 400 (400 MHz for ¹H; 100 MHz for ¹³C) spectrometer (Bruker, Rheinstetten, Germany). The chemical shifts are reported in parts per million (δ). The deuterium lock signal of the sample solvent was used as a reference, and the coupling constants (J) are given in hertz (Hz). The splitting pattern abbreviations are as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; and m, multiplet. The purity of all tested compounds was confirmed to be higher than 95% by analytical high-performance liquid chromatography (HPLC) analysis performed with a dual-pump Shimadzu LC-6AD system equipped with a VP-ODS C18 column (4.6 mm × 250 mm, 5 μm, Shimadzu).

To identify and characterize the naturally occurring hPL inhibitors in AGE, a UFLC system (Kyoto, Japan) coupling with a Triple TOF 5600 Mass Spectrometer system (Foster City, CA, USA) was performed to analyze predominant constituents. The chromatographic separation was employed by the Shimadzu VP-ODS C₁₈ column (2.0 mm × 250 mm, 4.6 μm) with a flow rate of 0.4 ml/min. The mobile phase was made up of water (0.1% formic acid) (A) and acetonitrile (B). The mobile phase program was shown in Supplementary Table S1. The column temperature was set at 40°C. The AGE sample (10 mg/ml) injection volume was 3 μl. The constituents were further analyzed via LC–TOF–MS/MS equipped with an electrospray ionization (ESI) source in negative ion mode. The MS parameters settings were shown in Supplementary Table S2.

Based on our previous results (Table S1), the samples were analyzed in chromatographic fingerprint by the Agilent 1260II HPLC-UV system equipped with a four-element pump (Santa Clara, CA, USA) with Shimadzu VP-ODS C18 column (150 × 4.6 mm, 5 μm, Kyoto, Japan). The column temperature was maintained at 30 °C. The analyte was eluted at a rate of 1 mL/min using a mobile phase consisting of 0.1% phosphoric acid aqueous solution and methanol under the following conditions as shown in Table 3.

The ACE inhibition rate was determined using HPLC. A Shimadzu LC‐2010A HT system (Shimadzu) equipped with VP‐ODS C₁₈ column and SPD‐M20A diode array detector with detection wavelength of 283 nm was used. The mobile phase consisted of methanol and water (3:7, v/v,) at a flow rate of 1.0 ml/min and a column temperature of 30°C. Sample solution, ACE, and HHL were prepared using BBS (0.1mol/L, pH 8.3) containing 0.3 mol/L KCl as a solvent. Sample solution (40 μl) was transferred to a test tube, and 20 μl of ACE and 100 μl of HHL (5 mol/L) were added. The mixture was incubated for 1 hr at 37°C, and HCl (1 mol/L) was used to stop the reaction. The treated sample solution (10 μl) was filtered through a 0.45 μm microporous membrane and then injected into the HPLC to determine the amount of Hip. The ACE scavenging capacity was determined as follows. $\text{Inhibition}(\%)=\left(A_0-A_1\right)/A_1\times 100\%$ where A₀ is the peak area of Hip in the blank group and A₁ is the peak area of Hip of the sample. Peak areas were determined by the software that came with the instrument.

A DOTA-c(NGR)₂ stock solution of 5 mg/mL was prepared in deionized water. A volume of 80 μL 1 mol/L HEPES (pH 5.0) was added to 200 μL ⁶⁸GaCl₃ (37-74 MBq) eluent, and then the mixture was incubated with 40 μL DOTA-c(NGR)₂ stock solution in a water bath at 95°C for 10 min. Quality control was performed by radio-HPLC (Agilent Technologies, Santa Clara, CA, USA) with a VP-ODS C18 column (Shimadzu, Kyoto, Japan) and an online radioactivity detector (Zonkia Scientific Instruments Co., Ltd., Anhui Province, China). The mobile phase was composed of solvent A, 0.1% trifluoroacetic acid (TFA) in water, and solvent B, 0.1% TFA in acetronitrile. The flow rate was set at 1 mL/min, and the UV wavelength was set at 220 nm. The mobile phase was gradiently changed from 20% solvent B to 30% solvent B in 20 min.
To evaluate the in vitro stability, ~3.7 MBq of ⁶⁸Ga-DOTA-c(NGR)₂ was incubated in phosphate-buffered saline (PBS) at room temperature or bovine serum at 37 °C, respectively. The radiochemical purity was determined at time points of 30 min and 1, 2 and 3 h.