Diamonsil c18 reverse phase column

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.

The drug loading and entrapment efficiency of the PTX-loaded micelles were determined as follows: 100 μL of PTX-loaded TS-GC micelles solution (concentration of TS-GC = 1.0 mg/mL) was centrifuged at 10,000 rpm for 10 min with ultrafilter (Vivaspin 500, MWCO 10 k, Sartorius Co., Göttingen, Germany). The unentrapped PTX amount in the ultrafiltrate (W₁) was determined using HPLC method. The HPLC system consisted of a mobile phase delivery pump (LC-10ATVP HPLC pump, Shimadzu, Kyoto, Japan) and a UV detector (SPD-10A UV/Vis detector, Shimadzu, Japan). For separation, a Diamonsil^TM C₁₈ reverse-phase column (200 × 4.6 mm, 5 μm, Dikma technologies Inc., Beijing, China) was used. The mobile phase consisted of a mixture of acetonitrile and water (60:40, v/v). The detector wavelength, column temperature, and flow rate of the mobile phase were set at 227 nm, 30 °C and 1.0 mL/min, respectively. The injection volume of the test samples was 20 µL.
In order to measure the total PTX content (W₀) in the micelles, another 100 μL of identical PTX-loaded micelles solution was ultrasonicated in 10 mL methanol to extract PTX from the micelles. The encapsulation efficiency (EE%) and drug loading (DL%) of PTX were calculated using the following Equations (1) and (2):

where W₁ is the PTX content in the ultrafiltrate, and W₀ is the total PTX amount in the solution. The unit was microgram.

The amount of PTX encapsulated into the micelles was determined as follows: a certain amount of lyophilized drug-loaded micelles was weighted, and then acetonitrile was added, followed by ultrasonication at 200 W for 10 min to extract PTX from the core of the micelles. After filtered through a 0.22 μm microporous filter, the PTX concentration was measured by HPLC method using a mobile phase delivery pump (LC-10ATVP, Shimadzu, Japan) and a Diamonsil^TM C₁₈ reverse-phase column (200 mm × 4.6 mm, 5 μm, Dikma Technologies Inc., Beijing, China). The mobile phase was the mixture of acetonitrile and water at a volume ratio of 70:30. The column temperature was set at 30 °C. The detection wavelength was 227 nm, and the flow rate was 1 mL/min. The drug loading (DL%) and drug encapsulation efficiency (EE%) were calculated with the following formulas.


where W_encapsulated, W_fed and W_micelles represented the weight of PTX encapsulated in the micelles, the PTX fed initially, and the weight of PTX-loaded micelles, respectively.