Dual ajs esi source

Metabolic flux analysis was performed on a 6550 Q-TOF mass spectrometer with Dual AJS ESI source (Agilent Corporation, MA, USA) equipped with a 1260 RRLC system (Agilent Co.).
LC separation was conducted under both acidic and basic conditions using a Waters BEH amide column (2.1 × 100 mm, 1.7 μm) at a column temperature of 35 °C. Mobile phase A was 95:5 acetonitrile: water with 10-mM ammonium acetate. Mobile phase B was H₂O with 10-mM ammonium acetate. Ammonium hydroxide (0.05%) and acetic acid (0.05%) were added in basic and acidic LC conditions, respectively.
The injection volume was 5 μL, and the flow rate was 0.25 mL/min. The basic gradient conditions were 0 min, 15% B; 12 min, 40% B; and 15 min, 47% B. The acidic gradient conditions were 0 min, 5% B; 5 min, 30% B; 6 min, 50% B; and 12 min, 50% B. The MS parameters were set as follows: Gas Temp, 200 °C; Drying Gas, 14 l/min; Nebulizer: 35 psig; sheath gas temp, 350 °C; sheath gas flow: 11 l/min; VCap, 3500 V; Nozzle Voltage, 600 V; Fragmentor, 380 V; and Octopole RF, 750 V. The data were acquired in a mass range from 70 to 1000 m/z in negative ion mode using MassHunter workstation (Agilent Co., MA, USA).

Commercially available reagents were used without further purification. Reactions were monitored by thin layer chromatography with silica gel 60 F₂₅₄ plates (E. Merck, Darmstadt, Germany). HPLC chromatography was carried out with the Autopurification system by Waters using fluoro-phenyl columns. ¹H and ¹³C NMR spectra were recorded on Bruker AC 200 (¹H: 200 MHz, ¹³C: 50 MHz), Bruker Avance Ultrashield 400 (¹H: 400 MHz, ¹³C: 101 MHz) or Bruker Avance IIIHD 600 spectrometer equipped with a Prodigy BBO cryo probe (¹H: 600 MHz, ¹³C: 151 MHz). Chemical shifts are reported in parts per million (ppm) and were calibrated using DMSO-d₆ as internal standard. Multiplicities are denoted by s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublet) and m (multiplet). Melting points were determined with a Büchi Melting Point B-545 apparatus. HR-MS was measured on an Aglient 6230 LC TOFMS mass spectrometer equipped with an Aglient Dual AJS ESI-Source.
Compounds 1 (PZ II 028), 2 (LAU156), 3 (LAU206) and 4 (LAU176) were synthesized and published previously^{23 (link)}. Synthesis of 5 (DCBS76) was conducted in analogy to previously outlined synthetic routes^{23 (link), 44 (link), 45 (link)}. The synthesis of 6 (DCBS96) was improved as described. Compound 7 (LAU462) was synthesized according to reported protocols^{23 (link), 46 (link), 47 (link)}.