Masshunter workstation data acquisition software

The effect of CH004 on the level of CTH in HepG2 cells was determined by a liquid chromatography-tandem mass spectrometry (LC/ESI-MS/MS). We followed a condition, which was previously established with LC/ESI-MS/MS^{56 (link)}, for quantification of CTH. The singly charged parent ion for CTH was observed at 223.1 m/z. The product ion scan showed dominant product ions at m/z values of 134 and 88.1. We selected the 223.1 → 88.1 transition for the quantitative method. Briefly, after the incubation with the compounds for 8 h in the presence of 100 μM Hcys and 100 μM Ser, the HepG2 cells were lysed with liquid nitrogen. 10 μl supernatant was injected to the Ultra Performance Liquid Chromatographic system on a Zorbax XDB C18 reverse phase column (4.6 × 150 mm, temperature-controlled at 10 °C), and eluted isogradiently with a solution mixed from acetonitrile (in the presence of 0.1% formic acid) and water (0.1% formic acid) (2:98, v/v) at a flow rate of 0.4 ml/min in a diode array detector (Agilent G4212A). The Agilent 6470 UHPLC-QqQ/MS system composes of a chomatographic system (Agilent 1260 Infinity) and a Triple Quad mass spectrometer fitted with an ESI source. Data was acquired in the MRM mode using Agilent MassHunter Workstation Data Acquisition Software (revision B.04).

Separation of STO-609 and its metabolites was achieved using a LC-QTOFMS system (Agilent Technologies, Santa Clara, CA) equipped with a 100 mm × 2.1 mm (Agilent XDB C18) column as previously described^{19 (link)}. Briefly, the column temperature was maintained at 45 °C. The flow rate was maintained at 0.3 ml/min with a gradient ranging from 2% to 98% aqueous acetonitrile containing 0.1% formic acid over a 15 min run. QTOFMS was operated in positive mode with electrospray ionization. Ultra high pure nitrogen was applied as the drying (12 L/min) and collision gas. The drying gas temperature was set at 325°C and nebulizer pressure was maintained at 35 psi. Capillary voltages were set at 3.5 kV. During mass spectrometry, real time mass correction and accurate mass were achieved by continuously measuring standard reference ions at m/z 121.0508, 922.0098 in the positive mode. The MS/MS of STO-609 and its metabolites was performed in targeted mode with a default isolation width of 4 m/z and collision energy ramp ranging from 20 to 50 V. Mass chromatograms and mass spectra were acquired by MassHunter® Workstation data Acquisition software (Agilent, Santa Clara, CA) in centroid and profile formats from m/z 100 to 1000. The acquisition rate was set as 1.5 spectra per second. Statistical analysis was conducted using Student’s independent t-test. Data are presented as mean ± s.e.m.