Metabolomic Analysis of TCA and Glycolysis

Tricarboxylic acid cycle and Glycolysis cycle metabolites were identified by using 5 mM ammonium acetate in water as buffer PH 9.9 (A) and 100% acetonitrile as a buffer (B) using Luna 3 µM NH2 100 A⁰ Chromatography column (Phenomenex, Torrance, CA). The Gradient used: 0–20 min-80% B (Flow rate 0.2 ml/min); 20–20.10 min- 80% to 2% B; 20.10–25 min-2% B(Flow rate 0.3 ml/min); 25–30 min 80% B (Flowrate 0.35 ml/min); 30–35 min-80%B (Flow rate 0.4 ml/min); 35–38 min 80% B (Flow rate 0.4 ml/min); followed by re-equilibration at the end of the gradient to the initial starting condition 80% B a Flow rate of 0.2 ml/min. All the identified metabolites were normalized by spiked internal standard (Mohammed et al., 2020c (link)).