Acquity csh c18 reversed phase column

Qualitative analyses of the sterols and sterol glucosides (including β-sitosterol-3-O-β-d-glucoside, campesterol-3-O-β-d-glucoside and cholesterol-3-O-β-d-glucoside) obtained from the in vitro MrSGT reactions were conducted by HPLC-tandem mass spectrometry (MS/MS; ThermoFinnigan, San Jose, CA, USA) as described before (Hoang et al. 2016 (link)). In brief, isocratic elution (methanol:acetonitrile:water:formic acid = 45:40:14.8:0.2 [v/v/v/v]) was performed on an Acquity CSH C₁₈ reversed-phase column (Waters, Milford, MA, USA; 2.1 × 50 mm, 1.7 μm) at a flow rate of 120 μL/min. The column effluent was introduced into the MS/MS (without splitting), which was operated in the positive ion mode. Acquisition was performed using MS/MS operated in the selective reaction monitoring (SRM) mode by choosing six different sets of mass transitions specific to both sterols and the corresponding sterol glucosides to detect the transition of the protonated precursor ion to the dominant product ion (415.5 [M+H]⁺ > 397.5 [M-H₂O+H]⁺ as a dehydrated product ion for β-sitosterol; 401.5 > 383.5 for campesterol; 387.5 > 369.5 for cholesterol; 577.5 > 399.5 [M-Glc+H]⁺ as an aglycone product ion for β-sitosterol-3-O-β-dglucoside; 563.5 > 385.5 for campesterol-3-O-β-d-glucoside; 549.5 > 371.5 for cholesterol-3-O-β-d-glucoside).

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.