Q exactive focus quadrupole orbitrap ms

The LC-MS/MS system consisted of a Thermo Scientific UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a HPG-3400RS Standard binary pump, WPS 3000 TRS analytical autosampler, and TCC-3000 SD Column compartment. The LC system was coupled with a high-resolution mass spectrometer (Q Exactive™ Focus quadrupole-Orbitrap MS; Thermo Fisher Scientific, Bremen, Germany). A heated electrospray ionization source II (HESI-II) probe was used as an ion generator with nitrogen used as the auxiliary, sheath and sweep gas. The mass spectrometry was operated in negative ion mode, with sheath gas, and the auxiliary gas was set to 45 and 10 aux units, respectively. The other parameters were set as follows: spray voltage to 2.5 KV, capillary temperature to 250 °C, S-lens RF level to 50, and Aux gas heater temperature to 400 °C. The LC method consisted of water (mobile phase A) and ACN (mobile phase B), both of which contained 0.1% formic acid, at a flow rate of 0.24 mL/min at 45 °C. For metabolic profiling, the gradient conditions were as follows: 15–57% of B at 0–21 min, 57–95% of B at 21–23 min, 5% of B at 23–25 min, 95–15% of B at 25–25.1 min, 15% of B at 25.1–30 min. The analytes were separated with a Kinetex^® C18 column (150 mm × 2.1 mm, 2.6 μm, Phenomenex, Torrance, CA, USA).

An HR-MS coupled with ultrahigh-performance liquid chromatography (UHPLC) detected the derivatized lactate and pyruvate. The UHPLC system Dionex Ultimate 3000 (Dionex Softron GmbH, Germering, Germany) was used. A reverse-phase liquid chromatography column, the 150 mm × 3 mm, 3-μm Shim-pack CIS C18 column (Shimadzu Corporation, Kyoto, Japan), at 40 °C temperature was used to separate the derivatized lactate and pyruvate. The gradient elution state for separation was as follows: 5% of B between 0 and 2 min; 5–65% of B between 2 and 10 min; 65–90% of B between 10 and 11 min; 90% of B between 11 and 13 min; 90–95% of B between 13 and 14 min; and 5% of B between 14 and 17 min. The MS detection was performed on Q-Exactive Focus quadrupole-Orbitrap MS (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated electrospray ionization (HESI-II) ion probe. The lactate and pyruvate were detected in positive ion mode after derivatization. Moreover, MS-grade solvents were used as the mobile phase with a flow rate of 400 L/min in the gradient elution mode. Water with 0.1% formic acid and 0.1% formic acid in ACN were solvent A and solvent B, respectively, for the LC–MS analysis.