Waters acquity uplc system

The BIAs in S. acutum were identified and quantified using the Waters ACQUITY Ultra-Performance Liquid Chromatography (UPLC) System (Waters, Milford, MA, United States) and the Q Exactive Plus Orbitrap Mass Spectrometer (Thermo Fisher Scientific., Waltham, MA, United States). Dried powder samples (0.25 g) of the root, leaf, stem, and seed of S. acutum were accurately weighted and subsequently extracted with 20 mL of methanol solution for 30 min in an ultrasonic bath. Chromatographic separation was carried out on the Waters ACQUITY UPLC system equipped with a BEH C18 column (2.1 × 100 mm, 1.7 µm), and the column temperature was kept at 30°C. The mobile phases were water with 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.3 mL/min. The solvent gradients for B were 0–6 min, 5%; 6–20 min, 5–16%; 20–22 min, 16–95%; 22 –25 min, 95%; and 25–26 min, 5%. The Q Exactive MS system used the electrospray ionization source (ESI) in the positive ion mode. The spray voltage and capillary temperature were 3.0 kV and 320 °C, respectively. The flow rates of the atomization gas and heating auxiliary gas were set at 35 arb and eight arb, respectively. The auxiliary gas heating temperature was set at 350°C.

Optical rotations were measured on a JASCO P-2000 polarimeter using a 1-cm cell. UV and electronic circular dichroism (ECD) spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics, Surrey, UK). 1D and 2D NMR spectra were obtained with Bruker AVANCE III HD 850 spectrometers (Bruker, Billerica, MA, USA) at the National Center for Interuniversity Research Facilities at Seoul National University (NCIRF). UHPLC-Q/TOF-MS analyses were performed on a Waters Acquity UPLC system (Waters Co., Milford, MA, USA) coupled with a Waters Xevo G2 QTOF mass spectrometer (Waters MS Technologies, Manchester, UK) that was equipped with an electrospray interface (ESI). The absolute configurations of the amino acids in compounds 1 and 2 were determined using an Agilent 6120 quadruple MSD consisting of a 1260 Infinity pump, a 1260 Infinity autosampler, a 1260 Infinity DAD (Agilent Technologies, Santa Clara, CA, USA), and an Agilent Zorbax SB-C₃ column (150 × 4.6 mm, 5 μm) at 50 °C. Semi-preparative HPLC separations were performed with a system consisting of a Gilson 321 Pump and a UV/Vis-151 detector (Gilson Inc., Middleton, WI, USA). Extra-pure grade solvents for extraction, fractionation, and isolation were purchased from Dae Jung Pure Chemical Engineering Co. Ltd., Siheung, Korea. Deuterated DMSO for NMR analyses was purchased from Merck (Darmstadt, Germany).

Each freeze‐dried leaf was ground to a fine homogeneous powder using a grinder (TissueLyser II, Qiagen, Germany) at 1800 rpm for 2 min. Five milligrams of leaf powder were suspended in 0.5 ml of 2% formic acid solution containing internal standard (heliotrine, 1 μg ml⁻¹). After centrifugation and filtration, 25 μl of the aliquot was diluted with 975 μl of 10 mM ammonium hydroxide solution; 10 μl of diluted aliquot was injected into a liquid chromatography‐tandem mass spectrometer (LC‐MS/MS) composed of a Waters Acquity UPLC system (Waters, Milford, MA, USA) coupled to a Waters Premier XE tandem mass spectrometer (Waters). The mass spectrometer was operated in positive electrospray mode and the samples were screened for a total of 45 PAs with output data processed using Masslynx 4.1 software (Waters). Detailed information on the LC‐MS/MS procedure is described in Cheng et al. (2011).

UPLC was performed on a Waters ACQUITY UPLC™ system (Waters Corp., Milford, MA, USA) equipped with a binary solvent delivery system, a UV detector, and an auto-sampler. Chromatographic separation was performed on a Waters ACQUITY BEH C18 column (i.d., 100 mm × 2.1 mm, 1.7 µm particle size; Waters Corp.) and the injection volume was 5 µL. The column temperature was set at 37 °C and the flow rate was 0.3 mL/min. The mobile phase consisted of 0.1% v/v formic acid in water (A) and 0.1% v/v formic acid in acetonitrile (B). The initial condition was 5% B for 1 min and linearly increased to 100% B over 9 min. Total run time was 14 min including re-equilibration of the column to the initial conditions. For MS experiments, the Waters Q–TOF Premier (Micromass MS Technologies, Manchester, UK) was operated in negative ion mode with an m/z range of 100–1000. The source temperature was set at 100 °C, the collision energy was set at 10 eV, the collision gas flow was 0.3 mL/min, and the desolvation gas was set to 650 L/h at a temperature of 300 °C. The capillary voltage and sample cone voltage were set at 2.5 kV and 50 V, respectively. The V mode was used for the mass spectrometer and data were collected in the centroid mode with a scan accumulation of 0.2 s. Leucine encephalin was used as reference lock mass (m/z 554.2615) by independent LockSpray interference.

Harmaline and harmine concentrations were simultaneous quantitative determined on a Waters-ACQUITY™ UPLC system (Waters Corp., Milford, MA, USA) using an ACQUITY UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm particle size). Mass spectrometric detection was performed using a triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA) equipped with electrospray ionization in positive ionization mode, and all other instrumental parameters were set according to our previous study (Li et al., 2016 (link)). The UPLC-ESI-MS/MS method was well-validated (data not shown) and the analytical method was successfully applied to determine the concentration of harmine and harmaline in the HBSS buffers. Figure S2 presents the representative of typical MRM chromatograms of blank HBSS, blank HBSS spiked with harmine, harmaline and IS, and IS-spiked HBSS sample collected at 30 min after administration of harmine and harmaline.