Lc 30a uhplc system

The purification and characterization of the active fractions followed the same procedure as previously described (47 (link)). Briefly, S. sonnei cells were cultured overnight in LB medium, and the pH of the supernatant was adjusted to 4 with hydrochloric acid and then extracted with an equal volume of ethyl acetate. After the extract was evaporated and concentrated, the residue was quantified and dissolved in methanol. The extract was analyzed by HPLC using a C₁₈ reverse-phase column (Atlantis T3 column, 5 μm, 4.6 mm by 250 mm) and eluted with methanol-water (from 5:95 to 70:30 vol/vol) at a flow rate of 1 mL/min. Finally, the active fractions were detected, concentrated, and further purified by HPLC using a semipreparative C₁₈ reverse-phase column. Peaks were monitored using a UV detector at 210 nm and 254 nm.
The ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded on an AVANCE III HD 400 (temperature, 298.0 K; Bruker, Billerica, MA, USA) operating at 400 MHz for ¹H or 101 MHz for ¹³C. Ultra-high-performance liquid chromatography (UHPLC)-ESI-MS/MS was performed in an LC-30A UHPLC system (Shimadzu Corporation, Kyoto, Japan) with a Waters C₁₈ column (1.8 μm, 150 by 2.1 mm) and a Shimadzu 8060 QQQ-MS mass spectrometer with an ESI source interface.

A. baumannii ATCC17978 cells were cultured overnight in LB medium, and the supernatant was extracted with an equal volume of ethyl acetate. After the ethyl acetate was concentrated by evaporation, the extract was dissolved in methanol and subjected to HPLC analysis on a C₁₈ reverse-phase column (Atlantis T3 Column, 5 μm, 4.6 mm × 250 mm) and then eluted with acetonitrile-water (from 10:90 to 70:30 vol/vol) at a flow rate of 1 mL/min. The active fractions were detected, concentrated, and purified by HPLC using a semipreparative C₁₈ reverse-phase column. Peaks were monitored using an UV detector at 210 nm.
The ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded on an AVANCE III HD 400 (Temperature 298.0 K, Bruker, Billerica, MA, USA) operating at 400 MHz for ¹H or 101 MHz for ¹³C. UHPLC-ESI-MS/MS was performed in a Shimadzu LC-30A UHPLC system (Shimadzu Corporation, Kyoto, Japan) with a Waters C₁₈ column (1.8 μm, 150 × 2.1 mm) and a Shimadzu 8060 QQQ-MS mass spectrometer with an ESI source interface (Shimadzu Corporation, Kyoto, Japan).

Bacteria were freshly inoculated and cultured overnight at 37°C with shaking, and then the bacterial liquid was diluted to an OD₆₀₀ of 0.05 in fresh medium and cultured overnight until the OD₆₀₀ reached 3.0. The supernatant of the bacterial liquid was collected by centrifugation and extracted with the same amount of ethyl acetate. The extract was evaporated, concentrated, and then dissolved in methanol. Finally, the extract was subjected to a Shimadzu LC-30A UHPLC system for quantitative analysis.