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Sample Preparation
Tryacylglycerols (TAGs … Sample Preparation
Tryacylglycerols (TAGs) in C57BL/6 mouse (purchased from SLC, Shizuoka, Japan) liver (100 mg) and white adipose tissue (WAT) (100 mg) were extracted using Bligh and Dyer's method in the following procedure: the liver and WAT were homogenized with 6 mL chloroform/methanol (1:2) for 10 strokes and left for 1 h at room temperature. Each phase separation was achieved by adding 2 mL chloroform and 2 mL water. After vortexing, the mixture was centrifuged at 3000 rpm for 10 min. The bottom organic layer containing the total lipid extract was collected and dried under a gentle stream of nitrogen, and then dissolved in chloroform / methanol (2:1) at a concentration of 10 ug (tissue weight) /uL.
LC conditions
Reverse-phased LC separation was achieved using ACQUITY UPLC BEH column (1.0 mm×150 mm i.d., particle size 1.7 um, Waters Corporation, Milford, MA, USA) at 45C. The mobile phase was acetonitrile / methanol / water : 19 / 19 / 2 (0.1% acetic acid + 0.028% ammonia) (A), and isopropanol (0.1% acetic acid + 0.028% ammonia) (B), and the composition was produced by mixing these solvents. The gradient consisted of holding solvent (A/B:90/10) for 5 min, then linearly converting to solvent (A/B:60/40) for 35 min and then linearly converting solvent (A/B:45/55) for 50 min. The mobile phase was pumped at a flow rate of 50 uL/min and the column pressure was adjusted lower than 5000 psi. Typically, 2 uL of sample solution was injected.
ESI-MS conditions
The ESI-MS analysis was performed using a quadrapole/time of flight hybrid mass spectrometer (QTOF microTM, Micromass, Manchester, UK) with an ACQUITY UPLC system (Waters Corporation). The mass range of the instrument was set at m/z 200–1100 and scan duration of MS and MS/MS at 0.5 s in positive ion mode. The capillary voltage was set at 3.5 kV, cone voltage at 30V, and source block temperature at 100C. The collision gas used for MS/MS experiments was argon (7.5×10E5 mbar), and the collision energy was set at 30 V. and the collision energy was set at 30 V.
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