1260 high performance liquid chromatography system

The enzymatic synthesis of AA-2G followed the method described in Tao et al. (2018) (link). Briefly, 0.5 g L-AA and β-CD were accurately weighed and dissolved into 5 ml water. Ten percent NaOH was added to adjust the pH to 5, and then, the recombinant enzyme solution was purified (enzyme solution was added according to the content of cyclodextrin) to generate AA-2G. Then, 10 g/L Na₂SO₃ was added to control oxygen. Finally, distilled water was added to 10 ml. With the above conditions unchanged (40°C and pH 5), various substrate types (α-CD, β-CD, γ-CD, and soluble starch), pH levels (3, 4, 5, 6, 7, and 8), temperatures (20, 30, 40, 50, and 60°C), enzyme concentrations (25, 50, 75, 100, and 125 U/g β-CD), donor concentrations (20, 30, 40, 50, and 60 g/L), and reaction times (6, 12, 18, 24, and 30 h) were tested to identify the optimal conditions for increasing the production of AA-2G.
An Agilent 1260 High Performance Liquid Chromatography (HPLC) system with kromasil 100-5C18 column (4.6 mm × 250 mm) was used to assess the quantity of AA-2G present in the reaction solution described above. In this work, the mobile phase was 20 mM H₃PO₄ at an 0.8-mL/min flow rate, the column temperature was 25°C, and the PDA detector used 242 nm. The peak area of the standard sample AA-2G was used to calculate the AA-2G yield.

The release of rapamycin from the PLGA nanoparticles was monitored in PBS (pH 7.4) containing 10% isopropanol at 37 °C under gentle shaking. In brief, 4 mL of PLGA nanoparticles (1 mg/mL) were transferred into a dialysis bag (10-kDa cutoff; Sigma-Aldrich) and immersed in 20 mL of PBS. At predetermined time intervals, 1 mL of release buffer was collected for measurement, and the same amount of fresh buffer was replenished. The concentration of released rapamycin was determined with an Agilent 1260 high-performance liquid chromatography system.