Drug EE% was determined
by the ultrafiltration centrifugation method.19 (link) Briefly, 1 mL of FH-HEX was centrifuged in an Amicon centrifugal
tube (100 000 molecular weight cutoff (MWCO), Millipore) at
15 000 rpm for 15 min at 4 °C. The supernatant was then
collected, and the amount of free FH was quantified using a previously
validated HPLC (Dionex, Thermo Scientific), equipped with an LPG-3400SD
quaternary pump, a WPS-3000TSL autosampler, a variable-wavelength
detector (VWD-3000), and a reverse-phase C18 column (Hypersil
ODS, 150 × 4.6 mm2, 5 μm). The wavelength of
the UV detector was set at 226 nm. The samples were separated using
acetonitrile and deionized water containing 10 mM aqueous triethylamine
at a ratio of 55:45 v/v at a flow rate of 1 mL/min at 25 °C.
The coefficient of determination (R2)
of the FH calibration curve in PBS (pH 7.4) in the concentration range
of 1–100 μg/mL was 0.9994, and the respective limits
of detection (LOD) and quantification (LOQ) were 0.5 and 1 μg/mL.
The coefficients of variation (CV)% ranged from 2.1 to 4.9%, and the
accuracy for FH determination was 1.5–4.6% with a mean% drug
recovery of 97.5 ± 1.16%.
The adsorbed amount of FH on
the Amicon centrifugal tube membrane was determined by filtering an
FH solution of the same concentration, and then the amount of free
FH in the filtrate was quantified.56 (link)EE% of FH was calculated using the following equation:
by the ultrafiltration centrifugation method.19 (link) Briefly, 1 mL of FH-HEX was centrifuged in an Amicon centrifugal
tube (100 000 molecular weight cutoff (MWCO), Millipore) at
15 000 rpm for 15 min at 4 °C. The supernatant was then
collected, and the amount of free FH was quantified using a previously
validated HPLC (Dionex, Thermo Scientific), equipped with an LPG-3400SD
quaternary pump, a WPS-3000TSL autosampler, a variable-wavelength
detector (VWD-3000), and a reverse-phase C18 column (Hypersil
ODS, 150 × 4.6 mm2, 5 μm). The wavelength of
the UV detector was set at 226 nm. The samples were separated using
acetonitrile and deionized water containing 10 mM aqueous triethylamine
at a ratio of 55:45 v/v at a flow rate of 1 mL/min at 25 °C.
The coefficient of determination (R2)
of the FH calibration curve in PBS (pH 7.4) in the concentration range
of 1–100 μg/mL was 0.9994, and the respective limits
of detection (LOD) and quantification (LOQ) were 0.5 and 1 μg/mL.
The coefficients of variation (CV)% ranged from 2.1 to 4.9%, and the
accuracy for FH determination was 1.5–4.6% with a mean% drug
recovery of 97.5 ± 1.16%.
The adsorbed amount of FH on
the Amicon centrifugal tube membrane was determined by filtering an
FH solution of the same concentration, and then the amount of free
FH in the filtrate was quantified.56 (link)EE% of FH was calculated using the following equation:
