LC–MS/MS analysis
LC instrumentation included a high-performance liquid chromatography integrated system (Thermo Fisher Scientific Inc., Waltham, MA, USA) with a XBridge BEH Amide column (2.5 μm, 2.1×100 mm, Waters) at 25°C. MS experiments were performed using a triple quadrupole mass spectrometer TSQ Quantum UltraTM (Thermo Fisher Scientific Inc., Waltham, MA, USA). The auto-sampler temperature was kept at 4°C. 5 μL of sample was injected with the full loop mode into the system. The XBridge BEH Amide column was used under acidic conditions, and the mobile phase consisted of two eluents: eluent A was 20 mmol/L NH4Ac in Milli-Q water, pH 6.5 and eluent B was 100% acetonitrile. A gradient was run at a flow rate of 0.3 mL/min; it started with 5% A, which was increased linearly to 60% A in 20 min and stayed at 60% A for 5 min, then decreased to 5% A in 10 min. An equilibration time of 15 min was allowed before the next injection.
Ionization was performed using an electrospray ionization source (ESI) operating in the positive and negative ionization modes. Sheath and auxiliary gas was nitrogen. The collision gas was argon. Xcalibur 2.2 software (Thermo Fisher Scientific Inc.) was used for instrumental control and data acquisition. Eighteen compounds presented greater ionization efficiency in negative than in positive ionization mode (namely G6P, F6P, FBP, 6PG, R5P, T6P, TRE, GAP, GOLP, 2+3PG, PEP, PYR, OAA, CIT, AKG, SUC, FUM and MAL), while for eight compounds the positive mode improved ionization efficiency (namely ATP, ADP, AMP, NAD+, NADH, NADP+, NADPH and AcCoA). Selected reaction monitoring mode was set, and the collision energy for an effective fragmentation of the selected precursor ions was optimized according to the analyte. The spray voltage, vaporizer temperature, sheath gas pressure, collision pressure, and aux gas pressure were 2.5 kV, 250 °C, 35 psi, 1.5 psi, and 10 psi, respectively.