Agilent 6410 triple quadrupole lc

MT921 was analyzed by modifying the protocol from a previously published paper, using an Agilent 6410 Triple Quadrupole LC-MS/MS system (Agilent, Wilmington, DE, USA) equipped with an Agilent 1200 series HPLC system [50 (link)]. MT921 was separated using an XBridge C18 column (2.1 mm × 100 mm, 3.5 µm; Waters, Milford, MA, USA). The mobile phases consisted of water and acetonitrile (40:60 υ/υ) with 0.1% formic acid at a flow rate of 0.2 mL/min. The retention times of MT921 and chenodeoxycholic acid (IS) were 2.1 min and 3.4 min, respectively. Quantitation was carried out using the multiple reaction monitoring (MRM) mode at m/z 407.5 → 407.5 (collision energy (CE) of 20 eV; negative ion mode) for MT921 and m/z 391.3 → 391.3 (CE of 25 eV; negative ion mode) for IS. The analytical data was processed by MassHunter software (version B.01.04).

For LC-ESI-MS analytics, the ethyl acetate extracts were dried in vacuo and resolved in 15 ml 40% aqueous acetonitrile + 0.1% formic acid and 4 µl were injected onto the UHPLC (ultrahigh performance liquid chromatography) column. LC-ESI-MS analytics were performed using an Agilent6410 Triple Quadrupole LC/MS system in negative ionization mode coupled to an UHPLC 1290 Infinity-Series (Agilent Technologies, Waldbronn, Germany). A VisionHT C18 Column 50 mm×2 mm, 1.5 µm (Grace, Grace GmbH & Co KG, Worms, Germany) was used for separation. Samples were analysed by linear gradient elution using H₂O + 0.1% formic acid as solvent A and acetonitrile + 0.1% formic acid as solvent B. The gradient was from 5% to 100% solvent B in 5 min with a 1.4 min isocratic elution at 100% for solvent B. Mass spectrometry parameters were optimized with commercial bacillibactin (EMC microcollections GmbH; Tübingen, Germany): fragmentor voltage: 80 V; cell accelerator voltage: 3 V; capillary voltage: 3000 V; nozzle voltage: 500 V. The optimized method was used for product ion scans (collision energy: 30 eV (collision-induced dissociation, CID)) with bacillibactin ([M-H]⁻  =  881 Da) as precursor ion.