Lct premier xe ms

To produce sufficient quantities of the two PSG derivatives for the purpose of elucidating their chemical structure, multiple scale-up reactions were carried out as follows; the molar concentrations of PSs, UDP-Glc and MrSGT enzyme were maintained in the proportions described above, while scaling up the total volume of the reaction mixture to 1 mL. At the end of the reaction, reaction mixtures from more than five batches were pooled and extracted by ethyl acetate partitioning. The solvent layer obtained was evaporated to dryness using a centrifugal evaporator (EYELA, Tokyo, Japan) set at 40 °C, and reconstituted in 5 mL of the mobile phase used for the HPLC–MS/MS analyses. The extracts were immediately loaded onto a reverse-phase C₈ cartridge of the CombiFlash Rf medium-pressure liquid chromatography (MPLC) system (Teledyne ISCO, Lincoln, NE, USA), and the flow rate was set at 8 mL/min. The eluents passing through a UV detector were all automatically fractionated over a 50-min running time. The fractions confirmed to contain the PSG derivative with a good purity (>97 %) by the tracing HPLC–MS/MS analyses, were pooled and freeze-dried. Their structures were further confirmed by Varian INOVA 500 nuclear magnetic resonance (NMR, Varian Inc., Palo Alto, CA, USA) spectroscopic analysis together with high resolution (HR) LCT-premier XE MS (Waters, Milford, MA, USA) analysis.

HPLC-LCQ ion-trap MS/MS (ThermoFinnigan, USA) was performed using both acetonitrile:methanol:water:formic acid (40:40:19.8:0.2 v/v/v/v) mixture as isocratic mobile phase at a flow rate of 150 µl/min, and Acquity CSH C₁₈ reversed-phase column (Waters, 50 × 1.0 mm, 1.7 µm; USA) as the stationary phase. The column effluent was introduced into the MS/MS operated in the positive ion mode. Acquisition was performed using MS/MS operated in the selective reaction monitoring mode by choosing a pair of mass transitions specific to phenoxodiol and the corresponding glycosides to detect the transition of the protonated precursor ion to the dominant product ion (241.2 [M+H]⁺ > 223.2 [M-H₂O+H]⁺ as a dehydrated product ion for phenoxodiol and 403.4 > 241.2 [M-Glc+H]⁺ as an aglycone product ion for phenoxodiol glycoside). In addition, both high-resolution (HR) LCT-premier XE MS (Waters) and Varian INOVA 500 nuclear magnetic resonance (NMR, Varian Inc., USA) were employed for the structural elucidation of two kinds of phenoxodiol glycosides.