Nexera lc 30 ad nexera x2 uhplc system

LC-MS/MS analysis was performed on a Sciex 5500 operated in multiple reaction monitoring (MRM) mode coupled with a UPLC Nexera LC-30 AD Nexera X2 UHPLC system (Shimadzu, Kyoto, Japan). Sphingolipids were chromatographically separated using a HALO HILIC column (4.6 x 150 mm, 2.7 µm). The mobile phases consisted of 100:0.5% acetonitrile: formic acid (volume for volume, MPA) and 50 mM ammonium formate in 60:40:0.5 water: methanol: formic acid (volume for volume, MPB). The run time for this method was 6 min starting with a linear gradient from 1% to 25% of MPB in 2.7 minutes, followed by a linear gradient to 40% MPB from 2.7 to 3.5 minutes, a linear gradient to 45% MPB from 3.5 to 4.0 min and a linear gradient to 1% of MPB from 4.0 to 4.1 min, and a re-equilibrium step of 1.9 min to the initial 1% MPB (flow rate 1.0 mL/min). The injection volume was 25 µL using a column temperature of 50°C. The sphingolipids and deuterium-labeled internal standards were detected using MRM with a positive turbo spray ion drive mode with the following parameters curtain gas (CUR) 20, collision gas (CAD) high, ion spray voltage (IS) 5500 V, temperature (TEM) 500, ion source gas 1 (GS1) 40, and ion source gas 2 (GS2) 60. The Q1/Q3 transitions, dwell time, declustering potential, collision energy, entrance potential, and CXP for each analyte are listed in Supplementary Table 3.

Analysis was performed on a Sciex QTRAP 6500+ coupled to a UPLC Nexera LC-30 AD Nexera X2 UHPLC system (Shimadzu, Kyoto, Japan).. SPBP was chromatographically separated using a Venusil XBP C18 (L) column (2.1 x 50 mm, 3 µM). The mobile phases consisted of 100:0.1 deionized water: formic acid (volume for volume, mobile phase A2; MPA2) and 100:0.1 acetonitrile: formic acid (volume for volume, mobile phase B2; MPB2). The run time for this method was 4.5 min starting with 60% of MPA2 for 1 min, followed by a linear gradient from 60% to 10% of MPA2 in 2.5 min, maintenance of 10% MPA2 for 0.5 minutes, and equilibrated at 60% MPA2 from 3.1 minutes, and a holding step for 1.4 min to the initial 60% MPA2 (flow rate 0.7 mL/min). The injection volume was 110 µL using a column temperature of 60°C. SPBP and SPBP-d7 were detected using MRM in positive ion mode with the following 3. The SPBP retention time was compared to the retention time for the deuterium-labeled internal standard SPBP-d7. The lower limit of quantification (LLOQ) was defined as the lowest concentration of analyte that could be measured with reproducibility of ± 25% and an accuracy of 75-125%.