Eclipse plus reversed phase c18 column

The ¹²C-/¹³C-labeled mixtures of each sample were analyzed using a 1290 ultra-high performance liquid chromatography instrument (UHPLC, Agilent) linked to a Bruker Impact II quadrupole time-of-flight (QTOF) mass spectrometer (Bruker, Billerica, MA) with electrospray ionization (ESI). The separation was performed using an Agilent Eclipse Plus reversed-phase C18 column (2.1 mm × 150 mm, 1.8 μm particle size). Mobile phase A was 0.1% (v/v) formic acid in water. Mobile phase B was 0.1% (v/v) formic acid in acetonitrile (ACN). The gradient for the separation was: t = 0 min, 25% B; t = 10 min, 99% B; t = 13 min, 99% B; t = 13.1 min, 25% B; t = 16 min, 25% B. All the samples were injected in random order. QC samples were injected every 10 sample runs to monitor instrument performance. The flow rate was 400 μL min⁻¹ and the sample injection volume was 2 μL. The column temperature was 40 °C. All MS spectra were collected at a mass scan range of m/z 220–1000 at a spectral acquisition rate of 1 Hz in positive ion mode.

Mixed samples were analysed by LC-MS (Agilent 1290 LC linked to Bruker Impact II QToF mass spectrometer) according to the conditions described previously [20 (link)]. Briefly, an Agilent eclipse plus reversed-phase C18 column (150 × 2.1 mm, 1.8 μm particle size) was used with mobile phases A (0.1% (v/v) formic acid in water) and B (0.1% (v/v) formic acid in acetonitrile) following a gradient of t = 0 min, 25% B; t = 10 min, 99% B; t = 13 min, 99% B; t = 13.1 min, 25% B; and t = 16 min, 25% B at a flow rate of 400 µL/min and a column oven temperature of 40 °C. QC samples were injected every 20 sample runs to monitor the instrument performance. Calibration data were used to check the retention time. All calibration peaks were well aligned. Their retention times were consistent for all calibration data, showing good RT stability for data acquisition.