Quanoptimize software

The test compound was optimized on a Waters
Acquity UPLC XEVO TQ-S microsystem (Waters Corporation) running in
the multiple reaction monitoring mode with negative or positive electrospray
ionization using QuanOptimize software (Waters Corporation).
The column used for chromatographic separation was a C18 BEH 1.7
μm column, with 0.5 mL/min flow rate; 5 μL sample injection
volume; mobile phase A: 5% acetonitrile and 0.1% formic acid in purified
water; mobile phase B: 0.1% formic acid in acetonitrile; general gradient:
1% to 90% (for B); 2 min total running time.

Glucose and lactate levels in the media were analyzed at time 0 and after 72 h using LC-MS/MS. An H-Class UPLC system and AQCUITY UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm particle size, from Waters) were used for the separation of glucose and lactate. UPLC separation was coupled with negative-mode ESI to a Waters Xevo TQ-S mass spectrometer operating in MRM mode. The LC parameters were as follows: autosampler temperature, 5°C; injection volume, 5 μl; column temperature, 50°C; and flow rate, 400 μl/min. The LC solvents were solvent A, 50 mM ammonium formate in water (pH 3) and solvent B, acetonitrile. Elution from the column was performed over 2 min with an isocratic gradient of 40% solvent A and 60% solvent B. The capillary voltage was 2.92 kV, and the cone voltage was 50 V. The flow rates of cone gas and desolvation gas were 150 and 600 L/h, respectively. The source temperature was 150°C, and the desolvation temperature was 500°C. Argon was used as collision gas at 1.5 mTorr. Collision energies and source cone potentials were optimized for each transition using Waters QuanOptimize software. Data were acquired using MassLynx 4.1 and QuanLynx software. The following equation was used to calculate metabolite consumption/excretion per 10⁶ cells per hour, denoted as α:

where

and X is the cell number and t is the time in hours.

Details of LC/MS/MS analysis are described in Mavangira et al., 2016 [25 (link)]. In short, the quantification of metabolites was accomplished on a Waters Xevo-TQ-S tandem quadrupole mass spectrometer using multiple reaction monitoring (MRM). Chromatography separation was performed with an Ascentis Express C18 HPLC column (10 cm × 2.1 mm; 2.7 μm particles, Sigma-Aldrich, St. Louis, MO, USA) at 50 °C, with the autosampler at 10 °C. Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile. Flow rate was fixed at 0.3 mL/ min. Liquid chromatography separation took 15 min per sample. MRM parameters including cone voltage, collision voltage, precursor ion, product ion, and dwell time were optimized based on Waters QuanOptimize software by flow injection of pure standard for each individual compound. Total IsoP concentrations were obtained by addition of IsoP detected in each sample type.

LC/MS/MS was used for the determination of total glutathione following extraction from 30 mg muscle homogenized in 400 μL deionized water (pH 7.0) containing N-ethylmaleimide. The protein precipitate was then reduced using Tris (2-carboxyethyl phosphine) and the extraction was repeated. An internal standard of 20 uM GSH ammonium salts D-5 (Toronto Research Chemicals, North York, ON, Canada) was added to all samples and standards. Chromatographic separation of the thiols was achieved using a Waters Acquity UPLC^® HSS T3 1.8 μM (2.1 × 100 mm) column (Waters Corp. Milford, MA, USA) with a gradient elution consisting of acetonitrile and deionized water with 0.1% formic acid. Multiple reaction monitoring, optimized using Waters QuanOptimize software, was used for detection of ions generated by glutathione in the MS/MS detector.