Strata c18 e55 m

WPE was separated on a reversed-phase column (Strata C18-E55 µm, 70 Å, 10 g/60 mL, Phenomenex, Inc., USA). The column was equilibrated with distilled water (three column volumes). Lyophilized WPE (28 mg) was separately loaded into the column and distilled water (three column volumes) was added to elute the organic acids. The eluate was used as the water-eluted fraction. Then, methanol (three column volumes) was added to obtain the methanol-eluted fraction. The two fractions were evaporated at reduced pressure and temperature (< 35 °C) and the extracts were lyophilized separately to yield 22 mg and 10 mg, respectively.

The cartridge (Strata^® C18-E 55 µm [70 Å, 20 g/60 mL, Giga Tubes], Phenomenex, Torrance, CA, USA) was equilibrated with 3 × 40 mL of methanol (MeOH), conditioned with 3 × 40 mL of water and then loaded with 150 mg of Drosera extract suspended in 5 mL of deionized (DI) water. After loading, a wash step was performed with 40 mL of DI water. To enrich compounds from the sundew extract into various fractions, four different concentrations of MeOH solutions (30%, 42%, 60% and 100%) were prepared for elution. The solutions were acidified with 0.1% anhydrous acetic acid. The extract was eluted with 3 × 40 mL of ascending MeOH concentrations, collecting one eluate for each MeOH concentration. Finally, a wash step was performed with 3 × 40 mL of isopropanol. The flow rate was 2 mL min^-1 by setting a vacuum. The fractions tested (42%, 60%, and 100% MeOH fractions of D. rotundifolia and D. intermedia) were concentrated under reduced pressure at 40 °C. Last solvent residues of the 42% MeOH fraction were removed by lyophilization. For the 60% and 100% fractions, final solvent residues were removed by evaporation at room temperature because lyophilization decreased the naphthoquinones concentration.

Scan type: full MS in the positive-and-negative mode (alternating); scan range, 69–1000 m/z; resolution, 70,000; AGC-target, 3E6; maximum injection time, 200 ms; sheath gas, 30; auxiliary gas, 10; sweep gas, 3; spray voltage, 3.6 kV (positive mode) or 2.5 kV (negative mode); capillary temperature, 320 °C; S-lens RF level, 55.0; and auxiliary gas heater temperature, 120 °C. Annotation and data evaluation: Peaks corresponding to the calculated monoisotopic masses (MIM ± H⁺ ± 2 mMU) were integrated using TraceFinder software (Thermo Scientific, Bremen, Germany). Materials: Ultrapure water was obtained from a Millipore water purification system (Milli-Q Merck Millipore, Darmstadt, Germany). HPLC–MS solvents, LC–MS NH₄OAc and lamivudine were purchased from Merck. RP18-SPE Columns: 50 mg Strata C18-E (55 µm) in 1 mL tubes (Phenomenex, Aschaffenburg, Germany). Sonifier: Branson Ultrasonics 250 equipped with a 13 mm-Disintegrator-Sonotrode (Thermo Scientific). LC/MS-system: Thermo Scientific Dionex UltiMate 3000 UHPLC system hyphenated with a Q Exactive mass spectrometer (QE-MS) equipped with a HESI probe (Thermo Scientific). Particle Filter: Javelin Filter with an ID of 2.1 mm (Thermo Scientific). UPLC-precolumn: SeQuant ZIC-HILIC column (5 μm particles, 20 × 2 mm) (Merck). UPLC column: SeQuant ZIC-HILIC column (3.5 μm particles, 100 × 2.1 mm) (Merck).