Dgu 20a3r degasser

Acetonic extracts (100 μg/ml) of plants were filtered through a 0.45 μm membrane filter before injection into the HPLC system. LC-ESI-MS analysis was performed using a LCMS-2020 quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) equipped with an electrospray ionization source (ESI) and operated in negative ionization mode. The temperature of quadrupole was DL: 275°C, heat block: 450°C. Mass spectrometer was coupled online with an ultra-fast liquid chromatography system that consisted of a LC-20AD XR binary pump system, SIL-20AC XR autosampler, CTO-20AC column oven and DGU-20A 3R degasser (Shimadzu, Kyoto, Japan). A DiscoVery BIO Wide Pore C18-5 (Thermo Electron, Dreieich, Germany) (15 cm x 4.6 mm, 5 μm) was applied for analysis. The mobile phase was composed of A (0.1% formic acid in H₂O, v/v) and B (0.1% formic acid in methanol, v/v) with a linear gradient elution: 0–14 min, 10% B; 14–24, 20% B, 27–37, 55% B, 37–45, 100% B, 45–50, 10% B. Re-equilibration duration was 5 minutes between individual runs. The flow rate of the mobile phase was 0.4 ml/min, the column temperature was maintained at 40°C and the injection volume was 5 μl. Spectra were monitored in mode SIM (Selected Ion Monitoring) and processed using Shimadzu LabSolutions LC-MS software.

ORBI quantification in the solubility test was performed by a stability-indicating method with high performance liquid chromatography as described by Casedey and collaborators [48 (link)]. The chromatographic separation was achieved using a Prominence model Shimadzu™ liquid chromatograph (Kyoto, Japan) with a DGU-20A 3R degasser, SIL-20AC HT autosampler, CTO-20A oven, LC-20AD pump, and SPD-M20A diode array detector. The following chromatographic parameters were used. A Shim-pack CLC-ODS Shimadzu column (Kyoto, Japan) (dimensions 250 mm × 4.6 mm diameter and 5.0 µm particle size) at 25 °C was the stationary phase. The mobile phase used was a solution of 5% (v/v) acetic acid:methanol (80:20 v/v) at a flow rate of 0.7 mL·min⁻¹. UV detection was performed at a wavelength of 290 nm, and the injection volume of the sample in the chromatographic system was 20 μL.
A calibration curve was prepared using a stock solution in the mobile phase of ORBI Sigma standard (previously dried for 4 h at 105 °C in an oven) at 5 concentrations in triplicate (5, 10, 15, 20, and 30 µg·mL⁻¹). The results were analysed by linear regression of the peak area versus concentration. The regression equation for the calibration curve was y = 134.377x − 3751 with a correlation coefficient of (r) = 0.9999.

The simultaneous determination of d- and l-enantiomers of serine and glutamate in samples was accomplished using high-performance liquid chromatography (HPLC) as previously described [21 (link)]. Briefly, the HPLC system consisted of DGU-20A3R degasser (Shimadzu, Kyoto, Japan), LC-20AD pump (Shimadzu, Kyoto, Japan), CTO-20AC column oven (Shimadzu, Kyoto, Japan), FP-4020 fluorescence detector (Jasco, Tokyo, Japan), and M510S auto-sampling injector (Eicom, Kyoto, Japan). d-Serine was identified based on retention time and a well-resolved peak and further confirmed by loss of the peak due to degradation by addition of DAO (2–5 units for tissue, plasma, saliva; 1 unit for dialysate).