Eluent a

A Shimadzu UHPLC Nexera X2 attached to a Shimadzu (Tokyo, Japan) LCMS-9030 QTOF with ESI source was used. Once samples were filtered (0.45 μm nylon filter, Sigma-Aldrich, St. Louis, MO, USA), betalains were separated using a Shim pack XR–ODS C18 column (150 × 2 mm, 2.2 μm particle size), at 7500 psi maximum working pressure and maximum flow rate of 0.4 mL/min, and using 1% formic acid in water (v/v, eluent A) and a mixture of acetonitrile/water/formic acid (80:19:1) (eluent B) (Sigma-Aldrich, St. Louis, MO, USA). The volume injection was 5 μL. The chromatographic method started with 100% of A, followed by a linear gradient from 0 to 20% of B in 35 min and then a linear gradient from 20 to 100% of B in 5 min [6 (link)]. To re-establish the initial conditions, a linear gradient from 100% of B to 100% of A was used for 10 min. The identification of the individual betalains was performed in positive mode using a sweeping range of m/z 100 to 1000 u. An amount of 4.5 mL/min of nitrogen was used as the drying gas, the temperature was set at 300 °C, the nebulizer gas flow was 3 L/min, the interface temperature was 526 °C, the sampling rate was 5 µ/s, the heater gas flow was 10 L/min, and the detector voltage was 0.20 kV.

Culture medium from the cells, which were grown to 80% confluency, was exchanged to DPBS supplemented with 25 mM glucose and 2 mM leucine ¹³C_6, and cells were incubated for 4 h. Every hour during the incubation, an aliquot of the medium was sampled. The 40 μL of collected medium fractions were mixed with acetonitrile in a ratio of 1:10. The mixtures were subsequently centrifuged at 10,000× g for 10 min, and clarified supernatants were used for the LC-MS analysis.
The polar compounds were separated on the Sequant ZiC-cHILIC 3 µm, 100 Å, 150 × 2.1 mm (MERCK) connected into a setup consisting of a liquid chromatography system LC NEXARA X2 (Shimadzu) coupled to mass spectrometer operating in IT-TOF mode (Shimadzu). For separation of internal standards and compounds in the medium, a mobile phase was used consisting of acetonitrile (eluent A; Sigma) and water with 100 mM ammonium acetate (eluent B; Sigma). The mobile phase was 10% B, which then increased to 60% over 20 min, afterward decreased back to 10% for the next 10 min. The flow rate was 500 µL/min. The obtained spectra were processed with Skyline 21.1 software (MacCross Lab Software).