Finnigan ltq orbitrap xl

Plant samples were extracted with methanol (final concentration 75% v/v, containing 25 µM of 7-hydroxy-5-methylflavone as an internal standard). After homogenizing the samples, centrifugation, and filtration, hydrophobic compounds in the filtrate were removed by absorption to a C18 silica column (MonoSpin C18, GL Science, Tokyo, Japan). Standard compounds for FsDatabase construction and identification of parsley peaks were dissolved in methanol. LC-MS analyses were performed using Agilent 1100 or 1200 systems (Agilent, Palo Alto, CA) coupled to a Finnigan LTQ-FT (Thermo Fisher Scientific) or a Finnigan LTQ-Orbitrap XL (Thermo Fisher Scientific). The binary raw data from Xcalibur (.raw) and their experimental metadata for plant samples are deposited at MassBase^{27 (link)} and Metabolonote³¹ , respectively. Their IDs and peak data are available at the KOMICS website^{27 (link)} (http://www.kazusa.or.jp/komics/software/FlavonoidSearch). The details of the extraction and analysis procedures were described in Supplementary Methods.

HPLC–MS was performed with an Agilent 1200 system (Agilent) coupled to a Finnigan LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA), which was equipped with an electrospray source operating in the negative-ionization mode. An
aliquot of the extracted sample (5 μL) was injected into an Ascentis Express C18 (2.7 µm, 150 × 4.6 mm; Sigma, St. Louis, MO) with mobile phases that consisted of 0.1% (v/v) aqueous formic acid (solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B). The gradient program was as follows: 30–60% solvent B for the first 48 min, 60–95% solvent B for the next 2 min, 95% solvent B for the next 5 min, and 30% solvent B for the last 5 min, with a flow rate of 0.5 mL/min. The column oven temperature was set at 40 °C. HPLC–MS analysis was performed using electrospray ionization (ESI) in negative-ionization mode. The full scan (m/z 800–2000) used a resolution of 60,000. MS/MS were acquired on a Top5 data-dependent mode with a parent list for saponins.

The ribonucleosides were separated using a Dionex Ultimate 3000 HPLC system on an RP-18 column (Synergi, 2.5 µm Fusion-RP C18 100 Å, 100 × 2 mm; Phenomenex^®, Torrance, CA, USA). Mobile phase A was 10 mM ammonium acetate and mobile phase B was 80% acetonitrile. The gradient began with 0% B and increased to 20% B over 10 min and to 80% by 12 min. After a 4 min hold at 80% B, the column returned to 100% A over 1 min. The column was re-equilibrated with 100% A for 8 min. The flow rate was 0.2 mL/min with a column temperature of 30 °C. For untargeted analysis, the slower flow rate resulted in better peak separation and thus mass spectra generation for individual compounds.
High-resolution mass spectra of precursor and product ions were recorded by a Thermo Finnigan LTQ Orbitrap XL operated in positive ionization mode with a capillary voltage of 20 V and temperature of 275 °C. Sheath gas flow was set to 5, and auxiliary gas was set to 35.