Pursuit xrs ultra c18 column

Acyl-CoA species from the reaction were first separated from high molecular weight components using 3000 Da cutoff spin columns, then equilibrated with two volumes of 150 mM ammonium acetate/acetate buffer pH 4.5 containing 4% acetonitrile before injection into the high-performance liquid chromatography (HPLC). Acyl-CoA standards were purchased from Sigma-Aldrich then made into 10 mM stock in water. A Beckman System Gold HPLC system with an Agilent Pursuit XRs Ultra C18 column were used to separate the acyl-CoA species using a 150 mM ammonium acetate/acetate buffer pH 4.5 and a 4–24% acetonitrile non-linear gradient at 1 ml/min for 45 min. The gradients of acetonitrile were: 0–5 min 4%, 5–10 min linear increases from 4% to 16%, 10–35 min linear increases from 16% to 24%, 35–38 min remain at 24%, 38–40 min decrease from 24% to 4%, 40–45 min remain at 4%. In this method, a series of mixture of CoA and Acyl-CoA standards were run to calibrate the separations. The approximate retention times were: CoA, 13.5 min; acetyl-CoA, 15.5 min; malonyl-CoA, 8 min; succinyl-CoA, 15 min; glutaryl-CoA, 16 min, and pimeloyl-CoA, 18.5 min.

UHPLC–MS/MS analyses were performed using a Waters Acquity UPLC^TM H–Class system (Waters, Manchester, UK). The mass spectrometer was a Xevo TQS tandem quadrupole mass spectrometer (Waters) equipped with StepWave ion guide and an orthogonal Z–spray electrospray ionization (ESI) source. Pursuit XRs Ultra C18 column (2.8 μm; 2 × 100 mm) (Agilent, CA, USA) was selected for the separation of compounds. Statgraphics Centurion XVI v.16.2 (Statpoint Technologies Inc., VA, USA) and Microsoft Excel 2013 v.15.0 (Microsoft, WA, USA) were used for the statistical treatment of data. MassLynx v.4.1 software was used for UHPLC-MS instrument control, peak detection and integration.