Lc 10

The levels of the reduced (GSH) and oxidized (GSSG) glutathione, cysteine and cystine in the incubation mixtures were determined using the RP-HPLC method of Dominic et al. [27 (link)] with modifications [28 (link)]. The samples were separated on a 4.6 mm × 250 mm Luna C18 (5 µm) column with a Phenomenex Security Guard column filled with the same packing material. The chromatographic system consisted of LC-10 Atvp Shimadzu Corp. pumps, four channel degassers, column oven, a Shimadzu SIL-10 Advp autosampler and a Shimadzu Corp. SIL-10 SPD-M10Avp-diode array detector; Lab Solution LC software was used to control system operation and facilitate data collection. The standard curves were generated in the supernatant obtained from cellular homogenates in the range from 13 to 75 nM of each compound per ml. All the standard curves generated for the analyte were linear in the investigated concertation range.

The quantification of CsA in the spray-dried powders was achieved by dissolving 20 mg of powder in 25 mL of water:acetonitrile 40:60. Six samples were prepared and analysed by HPLC. The drug content analysis was conducted after the powder preparation and during the stability study.
CsA was quantified using an HPLC (LC-10, Shimadzu, Kyoto, Japan) equipped with a UV–Vis detector, set at a wavelength of 230 nm and using the column Nova-Pak C18 (3.9 × 150 mm, 4 µm; Waters, Italy). The mobile phase was constituted by a mixture of 65% acetonitrile and 35% ultrapure water, acidified at 0.1% with trifluoroacetic acid. The column temperature was set at 65 °C and the flow rate was fixed at 1.6 mL/min. The injection volume was 10 µL, the run time of was 10 min and the retention time for the CsA was about 5 min. The method linearity was over the range 0.1–2 mg/mL.

Hydrocortisone at a final concentration of 0.1 and 0.3 μM was added to the culture medium of uterine epithelial cells (5×10⁵/well). After 2 h, the culture medium was discarded, and lysis buffer (10 mM Tris [pH 7.5], 10 μg/mL aprotinin, 10 μg/mL leupeptin, 0.2 mM PMSF, 0.1% sodium dodecyl sulfate) was added to the dish. The lysate was sonicated on ice. Fatty acids in the sample were fluorescently labeled with 9-anthryldiazomethane (Adam; Funakoshi Co. Ltd., Tokyo, Japan) according to following procedure. The same volume of 0.2% Adam/ethyl acetate was added to the sample dissolved in methanol after extraction using the Sep-Pack C18 cartridge column. The mixture was sealed in nitrogen gas and kept at room temperature in the dark overnight. After drying with nitrogen gas, the residue was dissolved in 100 μL of ethyl acetate/methanol (1:1). Reverse-Phase-HPLC (high performance liquid chromatography) (LC-10; Shimadzu, Japan) was performed with a Symmetry C18 column (4.6×250 mm, 5 μm) (Waters, Milford, MA, USA) in acetonitrile/water/phosphoric acid (7:3:0.01) at a flow rate of 1.0 mL/min. The excitation and emission wavelengths were 365 nm and 412 nm, respectively. Organic solvents and other reagents were purchased from FUJIFILM Wako Pure Chemical Corporation (Japan).

The HPLC system consisted of a LC-6A or LC-10 unit (Shimadzu, Kyoto, Japan) equipped with UV-visible detector and a 20-microliter sample loop. All chromatographic runs were performed at room temperature at a flow rate of 0.7 mL/min. The eluates were monitored at 254 nm. The dead time was determined by injecting 1,3,5-tri-tertbutylbenzene onto the column.