2695 liquid chromatography system

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.

The chemical profile of GZZSZTW extract was determined by high-performance lipid chromatography (HPLC). Standard chemicals (acteoside, epicatechin, icariin, rutin, naringin, protocatechuic acid and protocatechuic aldehyde) were purchased from National Institute for Food and Drug Control (Beijing, China). HPLC analysis of GZZSZTW extract was performed using a 2695 liquid chromatography system (Waters, USA) equipped with a reverse phase Symmetry C18 Column (Waters, USA). The mobile phase was a gradient elution system consisted of water containing 0.1% (v/v) formic acid (A) and acetonitrile (B) with a flow rate of 1 ml/min as following: 0–20 min, 95–85% A; 20–35 min, 85–75% A; 35–50 min, 75–55% A; 50–70 min, 55–10% A. The photodiode array (PDA) detector was set at 254 nm, and the on-line UV spectra were recorded in the range of 195–400 nm.

Column chromatography (CC) were conducted with Silica gel (200–300 mesh, Qingdao Marine Chemical, Inc.) and Lichroprep RP-18 (40–63 μm, Merck). Waters 2695 liquid chromatography system was applied to run Semipreparative HPLC with a YMC-Pack 10 mm × 250 mm column (Pro C18 RS). Thin-layer chromatography was carried out on precoated TLC plates (200–250 μm thickness, Silica gel 60 F₂₅₄, Qingdao Marine Chemical, Inc.). Bruker DRX-500 and Avance III-600 MHz spectrometers (Bruker, Zűrich, Switzerland) were used to record 1D and 2D NMR data with solvent signal as internal reference. ESIMS, HREIMS and HRESIMS were obtained from a Shimadzu LCMS-IT-TOF MS (Shimadzu, Kyoto, Japan), a Waters AutoSpec Premier P776 MS (Waters Corporation, Milford, USA) or an Agilent G6230 TOF MS (Agilent Technologies, Palo Alto, USA). Shimadzu IR-450 instrument was used to evaluate infrared spectra with KBr pellets. A JASCO P-1020 digital polarimeter was applied to test optical rotations, using MeOH as solvent. Quantitative-PCR (q-PCR) was conducted on ProFlex™ PCR system (Thermo Fisher, Shanghai, China).