Agilent 1100 hplc uv system

FTY720 was purchased from Kangbaotai Fine Chemical Co., Ltd. (Hubei, China, batch no. 20110612, purity: 99.4%), as was its reference substance. Ophthalmic solutions of various concentrations (0.1%, 0.2% and 0.5%) of FTY720 were prepared by a senior pharmacist (Chulong Huang) according to the standard protocols46 (link) of the Formulation Division of Zhongshan Ophthalmic Center (Guangzhou, China). Acetonitrile, ethanol, and trifluoroacetic acid (chromatographic grade) were purchased from Merck (Darmstadt, Germany).
HPLC was used to evaluate the concentrations of the prepared FTY720 solutions. The Agilent 1100 HPLC UV system consisted of an 1100 quaternary pump, a 100-position auto-sampler, and a variable wavelength UV-IVS detector, which was operated using the ChemStation software. Ultrapure water and methanol (both of chromatographic grade) were obtained from Honeywell International (New Jersey, USA). Chromatographic analyses were performed using a 5-μm Gemini C18 column (4.6 mm × 250 mm i.d., Phenomenex, USA). The chromatographic conditions used are listed in Table 2. The linearity, precision, accuracy, selectivity, stability, and limit of detection of this method were validated according to the guidelines of the FDA and the International Conference on Harmonization for all of the assayed matrices.

The firmness was measured using a texture analyser (TA.XT2i; Stable Micro Systems, https://www.stablemicrosystems.com) fitted with a 5‐mm flat probe. Each fruit was penetrated to 5 mm in depth at a speed rate of 0.5 mm sec⁻¹ and the maximum force developed during the test was recorded in Newton (N). Each fruit was measured twice at opposite sides of its equatorial zone. LC‐MS was performed using an Agilent 1100 HPLC/UV system (Agilent Technologies, https://www.agilent.com) with a reverse‐phase column (Luna 3u C18(2) 100A, 150 × 2 mm; Phenomenex, https://www.phenomenex.com) and connected to a Bruker esquire3000plus ion‐trap mass spectrometer (Bruker, https://www.bruker.com). LC‐MS analysis was performed according to the protocol described by Ring et al. (2013 (link)). The values were expressed as per mil (‰) equivalent internal standard per dry weight using biochanin as internal standard.

For activity screening assays to identify bioactive fractions, SEE were separated into a Zorbax SB-C18 (Agilent, Santa Clara, CA, USA) semi-preparative column (9.4 mm i.d. × 250 mm, 5 µm) connected to an Agilent 1100 HPLC-UV system (Agilent, Santa Clara, CA, USA) equipped with a fraction collector. The mobile phase system was the same as described for SSE characterization by RP-HPLC. Fractionation was performed using the following method: a linear gradient from 10% to 25% of solution B from 0 to 5 min at a flow rate of 1.5 mL/min, linear gradient to 30% of solvent B from 5 to 10 min at a flow rate of 1.5 mL/min, linear gradient to 50% of solvent B from 10 to 25 min at a flow rate of 2.5 mL/min, and a linear gradient to 100% of solvent B from 25 to 30 min at a flow rate of 3 mL/min. Total method time was 35 min. Fractions with same retention time were pooled and evaporated in a GeneVac EZ-2 series (Genevac Ltd., Ipswich, UK); protein quantification was performed as describe in the Recovery of Skin Secretions Extract section. Purity of fractions collected was assessed using HPLC and 10% SDS/Tricine-PAGE.