Aqcuity uplc beh amide column

The liquid chromatography–mass spectrometry was performed on a 1290 Infinity Binary UPLC coupled with a 6540 UHD Accurate-Mass Q-TOF (Agilent Technologies, Santa Clara, CA, USA) as described previously by Hanhineva et al.^{43 (link)} Briefly, a Zorbax Eclipse XDB-C18 column was used for the reversed-phase separation and an Aqcuity UPLC BEH amide column (Waters, Milford, MA, USA) for the HILIC separation. After each chromatographic separation, the ionization was carried out using jet stream electrospray ionization (ESI) in the positive and negative mode, yielding four data files per sample. The collision energies for the MS/MS analysis were chosen as 10, 20 and 40 V, for compatibility with the spectral databases.

Glucose and lactate levels in the media were analyzed at time 0 and after 72 h using LC-MS/MS. An H-Class UPLC system and AQCUITY UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm particle size, from Waters) were used for the separation of glucose and lactate. UPLC separation was coupled with negative-mode ESI to a Waters Xevo TQ-S mass spectrometer operating in MRM mode. The LC parameters were as follows: autosampler temperature, 5°C; injection volume, 5 μl; column temperature, 50°C; and flow rate, 400 μl/min. The LC solvents were solvent A, 50 mM ammonium formate in water (pH 3) and solvent B, acetonitrile. Elution from the column was performed over 2 min with an isocratic gradient of 40% solvent A and 60% solvent B. The capillary voltage was 2.92 kV, and the cone voltage was 50 V. The flow rates of cone gas and desolvation gas were 150 and 600 L/h, respectively. The source temperature was 150°C, and the desolvation temperature was 500°C. Argon was used as collision gas at 1.5 mTorr. Collision energies and source cone potentials were optimized for each transition using Waters QuanOptimize software. Data were acquired using MassLynx 4.1 and QuanLynx software. The following equation was used to calculate metabolite consumption/excretion per 10⁶ cells per hour, denoted as α:

where

and X is the cell number and t is the time in hours.