Waters 2489 tunable absorbance detector

The MG MPs (20 mg) and the free MG (20 mg) were put into small vials containing 2 mL of water, AGJ and AIJ, respectively and sealed, and then all the vials were put in the water bath of 37 °C, 40 rpm/min. After 48 h, the samples were taken out and centrifuged at 8000 rpm for 10 min, and then filtered through a 0.22 μm membrane filter. The supernatant obtained was used for HPLC detection to determine MG concentration. The HPLC detection was implemented by a Waters 1525-2489 high performance liquid chromatograph (Waters Corporation, Milford, MA, USA) consisting of a pump (Waters 1525 binary) and UV detector (Waters 2489 Tunable Absorbance Detector), which was equipped with a Diamonsil C18 reverse-phase column (4.6 × 250 mm, 5 μm, DIKMA, Beijing, China). The mobile phase, composed of acetonitrile and 0.1% phosphoric acid, was delivered at 1 mL/min. The drug was detected at 325 nm and the injection volume was 10 μL. The linear regression equation y = 13,866,374.4100x + 50,262.5833 (R² = 0.9998) was obtained with concentration of MG standard solution as abscissa and the absorbency as the y-coordinate.

HK was quantified by a Waters 1525-2489 high performance liquid chromatograph (Waters Corporation, Milford, MA, USA) consisting of a pump (Waters 1525 binary) and UV detector (Waters 2489 Tunable Absorbance Detector), which was equipped with a Diamonsil C₁₈ reverse-phase column (4.6 × 250 mm, 5 μm, DIKMA, Beijing, China). A methanol (55%), acetonitrile (20%), and water (25%) mixture was used as mobile phase. Other parameters of HPLC detection were as follows: A wavelength was 294 nm, the flow rate of the mobile phase was 1 mL/min, and the injection volume was 10 μL.
A standard solution of HK was prepared as follows: HK (10 mg) was accurately weighed and dissolved in 10 mL methanol solution, and then the solution was diluted appropriately to obtain solutions of 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.007813, and 0.003906 mg/mL. Using the concentration of HK standard solution as the abscissa and the absorbency as the y-coordinate, the linear chart was constructed, and the regression equation was Y = 11326308.72X − 8679 (R² = 0.9998). The retention time of the HK was about 10–11 min.