Drc 1000

Frozen samples were powdered using dry ice powder via a mill cutter (Tube Mill control and MT 40; IKA, Staufen, Germany) and were then lyophilized by a freeze-dryer (dry chamber, DRC-1000; freeze-drying instrument, FDU-2100; EYELA, Tokyo, Japan). Four milligrams of powdered samples were weighed (AP324W, Shimadzu Corporation, Kyoto, Japan) and placed into a 2-ml tube with 5-mm zirconia beads. One milliliter of extraction solvent with 0.1% (v/v) formic acid in 80% (v/v) methanol and internal standards (8.4 nM of lidocaine and 210 nM of 10-camphorsulfonic acid) was added into the tube, and the metabolites were extracted using a bead-shocker (Shake Master NEO, Biomedical Science, Tokyo, Japan) for 2 min at 1,000 rpm, followed by centrifugation at 9,100 � g for 1 min. The extracted solutions were evaporated using a liquid handling system (MicrolabSTARplus, Hamilton Company, Reno, NV, USA), and the residual extract was redissolved with LC–MS grade water (FUJIFILM Wako Pure Chemical Corporation) so that the final diluted solution would be 40-fold. The redissolved solution was filtered (MZHVN0W50; Merck Millipore, Darmstadt, Germany). One microliter of the solution including 100 ng of the sample was subjected to widely targeted metabolomics.