2.2. Instruments and conditions
A Dionex UltiMate 3000 HPLC system with a binary pump, an on-line degasser, etc. was employed for analysis. Separation was carried out by chromatography at 40 °C using a Hypersil GOLD C18 column (100 mm x 2.1 mm, 1.9 mm particle size) and C18 guard column. Acetonitrile-0.1% formic acid (25:75, v/v) was introduced at a flow rate of 0.2 ml/min. Moreover, 2 μl aliquots were added to the HPLC system for analysis.
In order to analyze MS data, a Thermo Scientific TSQ Quantum Access MAX triple stage quadrupole mass spectrometer was operated in negative ionization mode with an electrospray ionization source (ESI).The typical parameters of the ion source were as follows: sheath gas pressure (N2): 20 units; spray voltage: 3500 V; collision gas pressure (Ar): 1.5 mTorr; and scan width: 0.002; selected-reaction monitoring (SRM) with monitoring ion pairs was used to analyze the samples at m/z 359 → 161 for RA, 358 → 161 for A1, and 164 → 146 for the internal standard (IS). Every channel had a scanning dwell time of 0.1 s. Xcalibur 2.2 software was used in the centroid mode to acquire and process data.