Inertsil ods 4v

For quantification of glutathione, cells were incubated and treated under the same condition as metabolome analysis except that metabolites were extracted with 80% methanol. After removing the insoluble fractions by centrifugation, the supernatants were collected and dried using a SpeedVac at 30-45 C. Sample preparation was performed based on a previous study 59 . Briefly, TCEP solution was added to cell samples and incubated for 30 min at room temperature. After centrifugation, the supernatant was derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) for 60 min at 60 C. For Bio-thiols separation, Inertsil ODS-4V (3.0 3 250 mm, GL Sciences, Tokyo, Japan) was used as the stationary phase. Mobile phase was 100 mM acetate buffer, pH 4.0/MeOH (97.5/2.5, v/v) at a flow rate of 0.4 mL min -1 . The SBD-thiols were detected by fluorescence with excitation and emission wavelengths of 375 and 510 nm, respectively.

The HPLC system consisted of a pump (PU-980, Jasco, Tokyo, Japan), a ternary gradient unit (LG-1580-02, Jasco), a degasser (DG-980-50, Jasco), an autosampler (AS-1550, Jasco), an intelligent column thermostat (CO-1560, Jasco), and a fluorescence detector (RF-20A, Shimadzu, Kyoto, Japan). Inertsil ODS-4V (250 × 3.0 mm i.d., 5 μm, GL Sciences, Tokyo, Japan) was used as an analytical column. Chromato-Pro (Run Time Corporation, Kanagawa, Japan) software was used to analyze chromatograms. The column temperature was set at 40°C. Fluorescence detection was carried out at an emission wavelength of 530 nm with excitation at 470 nm. The NBDamino acids were separated under isocratic elution conditions using different mobile phases, at a flow rate of 0.6 mL/min.