2777c ultra

Liquid chromatography was performed using the 2777C ultra-performance liquid chromatography (UPLC) system (Waters, Manchester, UK) as previously described [18 (link)]. A high-resolution tandem mass spectrometer Xevo G2 XS QTOF (Waters) was used to detect metabolites eluted from the column. The Q-TOF was operated in both positive and negative ion modes. For positive ion mode, the capillary and sampling cone voltages were set at 3.0 kV and 40.0 V, respectively. For negative ion mode, the capillary and sampling cone voltages were set at 2.0 kV and 40.0 V, respectively. The mass spectrometry data were acquired by Centroid MSE mode. The TOF mass range ranged from 50 to 1200 Da and the scan time was 0.2 s. For the MS/MS detection, all precursors were fragmented using 20–40 eV, and the scan time was 0.2 s. During the acquisition, the LE signal was acquired every 3 s to calibrate the mass accuracy. Furthermore, a quality control (QC) sample (pooled aliquots from all analyzed samples) was analyzed every 6 samples and used to evaluate the consistency of the LC-MS during the whole acquisition.

For metabolite extraction, fresh twig samples from different Taxus species (25 mg each, n = 15) were transferred to 1.5-mL Eppendorf tubes, and 800 μL pre-cooled methanol/water (1:1, v/v) was added to the tube with two steel balls. All of the tubes were placed into a pre-cooled 48-well tube holder and ground using the 2010 Geno/Grinder (SPEX SamplePrep, Metuchen, NJ, USA) for 2 min at a rate of 1900 strokes/min. The homogenized samples were extracted in 0.5-mL of the pre-cooled chloroform/methanol/water (v:v:v, 1:3:1) extraction solvent by vortexing for 15 min at 4 °C in the dark and then ultrasonication for 5 min on ice. The samples were centrifuged at 13,000 g for 15 min at 4 °C, and 550 μL of the supernatants were collected. The extracts were vacuum-dried and resuspended in a 50% methanol solution. The prepared extracts were then loaded onto the auto-sampler of the 2777C ultra-performance liquid chromatography (UPLC) system (Waters, Herts, UK) at 4 °C.