Co 1560

Reverse-phase high-performance liquid chromatography (HPLC) was performed on a JASCO HPLC system (JASCO Corporation, Tokyo, Japan), consisting of a PU-1580 intelligent HPLC pump, an LG-1580-04 quaternary gradient unit, a UV-1575 intelligent UV/VIS detector, a PG-1580-54 4-line degasser, and a CO-1560 intelligent column thermostat. The BORWIN chromatographic system (JMBS Developments, Le Fontanil, France) was used for HPLC data analysis. Chromatographic separation was accomplished on a Phenomenex C18 reverse-phase column (4.6×250 cm, 5 μm, Phenomenex Inc., Torrance, CA, USA) at 30°C and was monitored at 280 nm. The linear gradient solvent system consisted of 0.5% acetic acid in water (solvent A) and 100% CH₃CN (solvent B), and was adjusted from 75% (solvent A):25% (solvent B) to 0% (solvent A):100% (solvent B) over 60 min at a flow rate of 0.5 mL/min.

The HPLC system consisted of a pump (PU-2080 Plus, JASCO, Tokyo, Japan), a column oven (CO-1560, JASCO), and a fluorescence detector (RF-20A, Shimadzu, Kyoto, Japan). An Inertsil Amide column (150 × 3.0 mm i.d., 5 μm, GL Sciences, Tokyo, Japan) was used to investigate TCEP reaction conditions. The mobile phase was acetonitrile-40 mM ammonium formate buffer (pH 3.0) (75/25, v/v), at a flow rate of 0.4 mL/min. For faster analysis of biothiols in mouse serum samples, a ZIC-HILIC column (150 × 2.1 mm i.d., 3.5 μm, Merck) was used. The mobile phase was acetonitrile-30 mM ammonium formate buffer (pH 3.4) (72/28, v/v) at a flow rate of 0.3 mL/min. The column temperature was set at 35 °C, and the SBD-thiols were detected based on fluorescence, with excitation and emission wavelengths of 375 and 510 nm, respectively. The chromatograms were analyzed using Chromato-Pro software (ver. 5.00, Run Time Corporation, Kanagawa, Japan).