6545 lc q tof mass spectrometer

Stock solution of compound 1 was prepared by dissolving 0.1 mg of sample in 200 μL MeOH. The solution was further diluted with MeOH, filtered through a 0.45 μm hydrophobic PTFE filter, and analyzed using LC/MS/MS, Agilent 1290 Infinity II series with a 6545 LC/Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The analysis was conducted by injecting 1 μL of the sample using an Agilent Eclipse Plus C₁₈ RRHD (1.8 μm, 2.1 × 50 mm) set at 30 °C. The mobile phase consisting of formic acid in H₂O (0.1% (v/v)) (A) and formic acid in acetonitrile (0.1% (v/v)) (B) was delivered at a flow rate of 0.3 mL/min by applying the following programmed gradient elution: 0–3.0 min, 10% (B); 3.0–10.0 min, 10–100% (B); 10.0–12.0 min, 100% (B); 12.0–15.0 min, 10% (B). The MS system was equipped with an ESI source and operated in both negative and positive ionization modes with a data acquisition range from m/z 100 to 600.

An in-situ extraction of each culture was done by adding 2 mL of ethyl acetate to each well of the previously grown cultures on the microbioreactor system. After incubation for 60 min at 190 rpm at room temperature, the organic phase containing secondary metabolites was collected, dried under N₂ airflow, and resuspended in 20 μL of methanol to generate the analytes for LC-QTOF-MS/MS and to conduct antimicrobial assays. Aliquots of each extract (1 μL) were analyzed by LC-QTOF-MS/MS, in a Agilent 1290 Infinity II UHPLC coupled to an Agilent 6545 LC/QTOF mass spectrometer with an orthogonal electrospray ionization (ESI) interface (Agilent Technologies, Waldbronn, Germany); using a Zorbax C8 RRHD 1.8 μm (2.1 × 50 mm) column, elution gradient of 2.50 min at 0.417 mL/min from isocratic 90% H2O/MeCN (acetonitrile) to 100% MeCN (with isocratic 0.1% formic acid modifier). MS/MS analysis was performed on the same instrument for ions detected in the full scan at an intensity above 1,000 counts at 10 scans/s, with an isolation width of ∼4 m/z using a fixed collision energy of 20 eV and a maximum of three selected precursors per cycle. Samples were injected (5 μL) using an autosampler refrigerated at 4°C.

The whole procedure of pigment extraction, identification and quantification is described in detail elsewhere [36 (link)]. Briefly, total pigments were extracted in acetone by disrupting roughly 20 mg lyophilized biomass between glass beads in a FastPrep-24 instrument (MP Biomedicals, Santa Ana, CA, USA). The acetonic extract was separated on a 1290 Infinity II LC System (Agilent Technologies, Santa Clara, CA, USA) equipped with an Acclaim C30, 3 µm, 2.1 mm × 100 mm column (Thermo Fisher Scientific, Waltham, MA, USA). The masses were detected by a 6545 LC/Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) operated in atmospheric pressure chemical ionization (APCI) positive mode. The pigments were separately identified by comparison of detected and theoretical masses in combination with characteristic spectral absorption of each substance in agreement to literature [51 (link)]. Carotenoid mono- and diesters were assigned to adonixanthin and astaxanthin according to the characteristic absorption spectra of the respective unesterified form [52 (link)]. Astaxanthin was quantified on a Vanquish Flex HPLC system with DAD at 450 nm (Thermo Fisher Scientific, Waltham, MA, USA) with standard calibration. All other carotenoids were reference to astaxanthin at 450 nm [36 (link)]. Chlorophyll a and b were analyzed photometrically [53 (link)].