HPLC-MS-based analysis of short-chain acyl-CoA

Procedure of preparing samples for HPLC-MS-based acyl-CoA analysis was identical to that described in the CE-MS section, except that cells or liver tissues were rinsed in PBS, and that ¹³C2:0-CoA (150 μg/l) and ¹³C3:0-CoA (200 μg/l) prepared in methanol were added as internal standards. Some 800 μl of the aqueous phase of each sample was collected after centrifugation at 15,000 g for 15 min at 4 °C and was lyophilized in a vacuum concentrator. Samples were then re-dissolved in 20 μl of ice-cold 20% methanol. Measurement of acetyl- and malonyl-CoAs was based on a previous study⁷⁷ using a QTRAP (SCIEX, QTRAP 6500 plus) mass spectrometer interfaced with a UPLC system (Waters, ACQUITY UPLC system). Some 5 μl of each sample were injected onto an Acquity HSS T3 column (2.1 × 50 mm, 1.7 μm, Waters) attached to an Acquity BEH C18 pre-column (2.1 × 5 mm, 1,7 μm, Waters). Mobile phases A and B were water and acetonitrile, respectively (both contains 10 mM ammonium acetate and 0.05% ammonium hydroxide). The column temperature was maintained at 30 °C and autosampler temperature at 8 °C. The gradients were as follows: t = 0 min, 3% B; t = 1 min, 3% B; t = 13 min, 15% B; t = 14.1 min, 100% B; t = 14.7 min, 100% B; t = 14.71 min, 3% B; t = 17 min, 3% B. Mass spectrometry was in positive mode with multiple reactions monitoring mode (MRM), declustering potentials (DP) and collision energies (CE) set at 200 and 38 V, respectively (optimized using ¹³C2:0- and ¹³C3:0-CoA). The following transitions and retention time were used for monitoring each compound: 812.0, 305.3, and 2.2 min for ¹³C2:0-CoA, 810.1, 303.1, and 2.2 min for acetyl-CoA, 856.9, 350.2, and 0.8 min for ¹³C3:0-CoA, and 854.1, 347.1, and 0.8 min for malonyl-CoA. It should be noted that acyl-CoAs were extremely easy to degrade even frozen at −80 °C, so no pause was taken during the whole analytic process and no more than 6 samples were re-dissolved and kept in autosampler at one time.