Lc 2010a ht hplc

The dissolution test of both F₁ and F₂ tablets were evaluated on a Hanson Elite Vision G2 dissolution test (Hanson Research, Chatsworth, CA, USA) system using the USP dissolution apparatus 2, in 900 mL of HCl 0.1 N as the medium was kept at 37 °C, with a stirring rate of 75 rpm for 30 min. Then, the solutions were filtered using a 0.45 µm membrane filters and analyzed by HPLC to determine drug concentration. The OLM and HCT content were quantified using a previously developed and validated in-house HPLC method (not reported) consisting of a Shimadzu LC-2010A HT HPLC (Shimadzu, Tokyo, Japan) system, equipped with variable wavelength detector, pump, variable temperature compartment column and autosampler. The mobile phase was composed of ammonium phosphate adjusted to pH 3.0 with triethylamine (A) and acetonitrile (B) using a gradient elution method starting at A:B 74:26 to reach 60:40 in 13 min, a flow rate of 1.3 mL/min, 30 µL injection volume, detection at 230 nm in a Phenyl-Hexyl column (Agilent, Santa Clara, CA, USA) (250 mm × 4.6 mm, 5 µm), and a temperature of 30 °C.

Carotenoid were extracted from fresh petals at the flowering stage and detected following the methods of Cao et al.^{51 (link)}. Carotenoid analysis was performed using LC-2010AHT HPLC (Shimadzu, Kyoto, Japan) with C30 column (YMC, Kyoto, Japan). Carotenoids were identified by the typical retention time of the standard compounds, including violaxanthin (Sigma-Aldrich, Saint Louis, America), lutein (Solarbio, Beijing, China), α-carotene and β-carotene (Wako, Osaka, Japan). The identification of prolycopene was performed based on reported the typical retention time and relative order of carotenoid compound peaks^{22 (link),43 (link),51 (link)}. Carotenoid content was quantified according to Morris’ method^{52 (link)}. The total carotenoid content was the sum of all the detected carotenoid compound contents. Three biological replicates were used for all analyses and the calculation of means and standard deviations were conducted. The significant difference between 92S105 and 15S1040 was analyzed by t-test.