Accelatm u hplc system

The chromatographic separation was performed using an Accela^TM U-HPLC system (Thermo Fisher Scientific, San Jose, CA, USA) comprising of a UHPLC pump and a PDA detector and the samples were scanned from 200 to 400 nm. The MS analysis was performed using a the LTQ Orbitrap XL hybrid instrument (Thermo Fisher Scientific, San Jose, CA, USA).

This analysis was done to characterize the compounds presence in the EBN extract. Approximately 2 mg of EBN extract was dissolved in 1 ml of HPLC grade methanol. The solution was sonicated for 15 min and filtered through a membrane filter with pore size 0.22 μm before analysis. About 10 μL of the sample was injected into the AccelaTM UHPLC System (Thermo Scientific, San Jose, United States) equipped with quaternary pump, a build degasser, a PDA detector and an auto-sampler. Separation of the EBN sample was carried out by using A Luna Kinetex RP C18 column (2.6 μm, 2.1 mm I.D. x 150 mm) at a flow rate of 200 μL/min over 40 min by using acetonitrile: 0.1% formic acid in water as eluent. The step gradient and isocratic solvent composition was depicted in Supplementary Table S1.
The sample was analyzed by LTQ mass spectrometer (Thermo Scientific, San Jose, United States) with m/z spectrum ranged from 50 to 1000 was recorded in negative ionization mode. The setting of electrospray ionization modes was as follows: source accelerating voltage, 4.0 kV; capillary temperature, 350°C; sheath gas flow, 40 arb and auxiliary gas, 20 arb.
A reference standard of 18 types of amino acid (AAS18, analytical standard) was obtained from Sigma-Aldrich and used in this analysis. This reference standard was separated and analyzed under the same setting explained above.