Dionex u3000 hplc system

The samples were analyzed by Dionex U3000 HPLC system (Thermo Fisher), equipped with a pump and a CORONA Ultra Detector. An Eclipse XDB C18 column (150 mm × 4.6 mm I.D., 5 μm, Agilent) was utilized at a column temperature of 30°C. The mobile phase consisted of acetonitrile–methanol–water (65:10:25, v/v) containing 0.08% triethylamine with a flow rate of 1.0 ml/min. The evaporation temperature for detector was set at 30°C. Nitrogen was used as the carrier gas at a flow rate of 0.2 ml/min with the pressure of a nebulizing gas of 0.4 MPa. An aliquot (20 μl) of sample was injected.

The HPLC analyses were carried out on the Dionex U3000 HPLC system (Thermo Scientific, Waltham, MA, USA) equipped with a pumping device, an autosampler, a column oven, and a diode array detector. The quaternary solvent delivery pump was able to work up to a pressure of 600 bars. The columns evaluated were J’sphere ODS-H80 (150 × 4.6 mm, 5 µm) from YMC CO., LTD (Kyoto, Japan), Symmetry C18 (150 × 3.0 mm, 5 µm), and XTerra RP18 (50 × 3.0 mm, 3.5 µm) both from Waters (Milford, MA, USA). The column temperature was maintained at 30 °C and UV detection performed at 210 nm. UV spectra from 200 nm to 400 nm were recorded for peak identification. The injection volume was 10 µL. An isocratic mobile phase containing EtOH 96% and 10 mM of acetic acid aqueous solution, pH 3.35 (60:40, v/v) was used at a flow rate of 0.5 mL/min. The quality of separation between artemether and lumefantrine was evaluated in different proportions of solvents and for each condition, retention factors (k), resolutions (Rs), and symmetry factors were calculated. The best conditions were achieved using the Symmetry C18 column and a mobile phase composed of EtOH 96% and 10 mM of acetic acid aqueous solution, pH 3.35 (63:37, v/v).

Mass-spectrometric analysis of PA-glycans was performed by positive-ion mode ESI-MS on an LTQ XL linear ion trap mass spectrometer coupled to a Dionex U3000 HPLC system (Thermo Scientific, San Jose, CA). The fractions isolated through reversed-phase HPLC were trapped on a Hypercarb guard cartridge column (1 × 10 mm; Thermo Scientific, Waltham, MA) for enrichment and separation from contaminants. The samples were eluted with 0.1% (v/v) formic acid in water and 0.5% formic acid in acetonitrile. The flow rate was 50 µl/min, and the gradient conditions were varied for different samples. MS² analyses of PA-glycans were carried out by collision-induced dissociation in a data-dependent mode or with manually selected parent ion isolation. The peak intensities were extracted in the Mass+ + software ver. 2 (Shimadzu, Kyoto, Japan). All the MS and MS² spectra are displayed in Supplementary Fig. S2.
Sample preparation and mass-spectrometric analyses described above were performed in accordance with MIRAGE guidelines^{55 (link),56 (link)}. Details of the HPLC, MS, and MS² settings are given in Supplementary Table S8.