Guard column

After the addition of nonadecanoyl-LPC as an internal standard, plasma LPC was extracted using the Bligh and Dyer method [24 (link)]. The extract was dried under reduced pressure and reconstituted with methanol. After filtration using a 0.2-μm polytetrafluoroethylene membrane (Millex-LG; Millipore Corporation, Billerica, MA, USA), the filtrates were injected into the HPLC (Prominence; SHIMADZU CORPORATION, Kyoto, Japan) combined with an evaporative light scattering detector (ELSD-LTII, SHIMADZU CORPORATION). A Purospher^® STAR RP-18 endcapped (5 µm) LiChroCART^® (250 × 4 mm) was used as a separate column with a guard column (Merck KGaA, Darmstadt, Germany). The mobile phases were 75% acetonitrile with 0.1% formic acid (A) and 2-propanol (B), and the gradient conditions were: A:B = 100:0 for 15 min (1.0 mL/min), changed to 0:100 over 5 min, maintained at 0:100 for 15 min, then changed to 100:0 over 5 min (0.6 mL/min), and maintained at 100:0 for 10 min (1.0 mL/min). The settings for the detector were 40 °C, 350 kPa, and a gain of 12. The detected areas for different concentrations of palmitoyl-LPC standards were converted to a logarithmic scale to generate a fitting curve, and palmitoyl-, stearoyl-, oleoyl-, linoleoyl-, and linolenoyl-LPCs were quantified.

Metabolites were extracted from mid-exponential-phase culture pellets (five replicates) with a cold methanol method (40 (link)) and analyzed using an Agilent 6550 Q-TOF apparatus (Agilent Technologies, Santa Clara, CA). Liquid chromatography (LC) separation was conducted on an Agilent 1290 ultrahigh-performance LC (UHPLC) system (Agilent Technologies, Santa Clara, CA) with a 150- by 2.1-mm, 5-µm, 200-Å SeQuant ZIC-pHILIC column (EMD Millipore, Billerica, MA) and guard column of 20 by 2.1 mm, 5 µm (EMD Millipore, Billerica, MA) (41 (link)). MassHunter (Agilent) was used for initial data analysis, and Metabolite Atlas was used for targeted data analysis. Compounds were confirmed using authentic standards.

Cells were plated in triplicate plus an extra well for cell counting and cultured in standard medium for 18 h. Metabolite extraction buffer (MEB) was prepared composing of 50% methanol and 30% acetonitrile in water and kept at 4 °C. The counting well was then counted using the Invitrogen™ Countess™ automated cell counter based on manufacturer’s instructions to give the total number of cells per well. The media from the replicate wells was removed and the cells were washed once with 1xPBS. Metabolites were then extracted by adding 1 mL MEB per million cells and quickly scraped. The insoluble material was immediately pelleted in a cooled centrifuge (4 °C) at 13,000 rpm for 15 min and the supernatant was collected for subsequent LC–MS analysis. A HILIC column (4.6 × 150 mm, guard column 2.1 × 20 mm, Merck) was used for LC separation. The aqueous mobile phase solvent used was 0.1% formic acid in water (solvent A) and the organic mobile phase was 0.1% formic acid in acetonitrile (solvent B). The flow rate was set at 300 μl/min and the column oven set to 30 °C. The mobile phase gradient was described previously^{4 (link)}. Subsequent analysis was performed using the Xcalibur Quan Browser software (Thermo Fisher Scientific).