Quantitative Analysis of STB by HPLC

STB was quantitatively assayed by high-performance liquid chromatography (HPLC) according to the previous literature (Blanchet et al., 2009 (link); Iqbal et al., 2014 (link)), and the flow rate and column were adjusted to be more suitable for the detection of STB. Briefly, HPLC (Waters 2,695 liquid chromatography system) (Milford, MA, United States) was used in the formulation of CCD-RSM, a short-term storage stability study, an in vitro drug release study and an in vivo ocular pharmacokinetics study. An X-Bridge C18 column (3.5 μm, 3.0 × 150 mm) was used with a column temperature of 40°C. The mobile phase consisted of ammonium acetate buffer (0.02 mol/L) and acetonitrile (62: 38, V/V) at a flow rate of 0.4 mL/min. Twenty microliters of sample was injected, and the ultraviolet (UV) detection wavelength was set at 430 nm. The limit of quantitation (LOQ) of STB for HPLC was 25 ng/mL. The concentration of STB was calculated from the standard curve. The standard curve referred to the curve containing different concentrations of STB diluted with methanol. The quantitative analysis methods of STB followed the ICH guidelines, details of which are reported in the Supplementary Section S2.