Amazon x

Samples were analyzed in a Shimadzu Prominence liquid chromatography system with two Shimadzu LC-20ad (SIL-20a HT) (Shimadzu Corp., Quioto, Japan) automated injection pumps. Phenomenex Luna C18 column (250 × 4.6 mm − 5 μm) (Phenomex Inc., Torrance, CA, USA) was used to separate the components. The elution solvents used were A (0.1% HCOOH in acidified water) and B (0.1% HCOOH in acidified metanol/HPLC grade)—at a flow rate of 1.0 mL/min. Samples are eluted in a gradient system: 95% A/5% B-0 min-0% A/100% B-60 min and 100% B in 10 min with a runtime of 70 min. The liquid chromatogram was performed using electrospray ionization (ESI), multistage fragmentation (MSn) at an ion trap (IT) interface (Amazon X, Bruker, MA, USA) in the negative and positive mode under the following conditions: capillary voltage of 5 kV, capillary temperature of 325 °C, carrier gas flow (N₂) of 12 L/min, and nitrogen nebulizer pressure at 10 psi. Full scan analysis was recorded in the M/Z range from 100–1500 m/z, with two or more events.

The methanol extracts obtained above (1 mL) were centrifuged at 17,000 × g for 20 min. Twenty μL of crude extracts were diluted in 180 μL methanol before analysis, 5 μL of which was injected and analyzed by electrospray ionization–HPLC-MS by a Dionex UltiMate 3000 system coupled to a Bruker AmaZon X mass spectrometer with an ACQUITY UPLC™ BEH C18 column (130 Å, 2.1 × 100 mm², 1.7 µm particle size, Waters GmbH) at a flow rate of 0.6 mL min⁻¹ using acetonitrile (ACN) and water containing 0.1% formic acid (v/v) in a gradient ranging from 5 to 95% of ACN over 16 min. BPC spectra for RXPs were recorded in positive ion mode with the range from 80 to 1600 m/z and ultraviolet (UV) at 200–600 nm.

Isocratic elution method was applied for products analysis of luteolin homo-coupling. DI water with 0.1% formic acid was choose as mobile phase A while acetonitrile with 0.1% formic acid was applied for mobile phase B. The column was equilibrated with 71.5% mobile phase A for 10 min before isocratic elution of the same percentage mobile phase A from 0 to 35 min at a flow rate of 1.0 mL/min, 10 μL from reaction solution was injected into the HPLC system for analysis.
The LC–MS system was equipped with a C18 column (Phenomenex, Luna 5u C18, 250 × 4.6 mm) guard column (4 × 3.0 mm) was applied for product characterizations. The samples (10 μL) were filtered through 0.2 μm membrane (Merck Millipore, USA) before being injected into the HPLC system. The Waters 2998 Photodiode Array (PDA) Detector was connected to the HPLC system with detection wavelengths from 190 and 800 nm. Bruker AmaZon-X is applied for LCMS and LCMSMS for characterization and fragments analysis of unknowns. The LC conditions for LC−MS analysis were similar to those mentioned above. All mass spectra were acquired in both positive and negative ion modes using electrospray ionization. The parent ion was selected with a width of ±2.5 Da and fragmented with 50% setting.

A Shimadzu^® (Kyoto, Japan) High Performance Liquid Chromatography System, coupled with an Amazon X or micrOTOF II (Bruker Daltonics, Billerica, MA, USA) with an electrospray ion (ESI) source, was used to perform the ESI-MSⁿ and HRESIMS analysis, respectively. The LC System consisted of a LC-20AD solvent pump unit (flow rate of 600 μL.min⁻¹); a DGU-20A₅ online degasser; a CBM-20A system controller and a SPD-M20A (190–800 nm) diode array detector. The LC separation was performed on a Kromasil C-18 5 μm 100Å, 250 × 4.6 mm (Kromasil, Bohus, Sweden) analytical column. Injections (20 μL) were performed using an autosampler (SIL-10AF). The mobile phase consisted of 0.1% formic acid in water (solvent A) and methanol (solvent B). Exploratory linear gradient (5 × 100% B) was performed to elution in 90 min. The analysis parameters are as follows: capillary 4.5 kV, ESI in negative mode, final plate offset 500 V, 40 psi nebulizer, dry gas (N₂) with flow rate of 8 mL/min and a temperature of 300 °C. CID fragmentation, in Amazon X, was achieved in auto MS/MS mode using enhanced resolution mode for MS and MS/MS mode. The spectra (m/z 50–1000) were recorded every 2 s.

Organic extracts for each isolate and medium blank were prepared at a concentration of 1 mg/mL in MeOH. Liquid chromatography-mass spectrometry experiments were carried out in an HPLC LaChrom (VWR HITACHI-ELITE) coupled to ESI-IT mass spectrometer Amazon X (Bruker-Daltonics). The chromatographic analysis used a XTerra C-18 column (4.6 × 150 mm, 5μm) and a flow rate of 1.0 mL/min. The mobile phase consisted of 0.075% formic acid (A) and acetonitrile with 0.075% formic acid (B). The solvent program started with 10% B for 3 min and a linear increase to 40% B in 17 min, followed by a linear increase to 100% B in 15 min, then followed by 10 min at 100% B. The total analysis time for each sample was 50 min. The sample (20 μL) was injected by an autosampler. The mass spectrometry was carried out in both positive and negative ionization modes with a spray voltage at 4 kV and capillary temperature at 250°C. The mass range was set from m/z 70–3000. Raw data files generated by HPLC-MS were first converted to netCDF files.

All work with phosphines 1 and 2 was performed in a dry argon atmosphere using the standard Schlenk vacuum technique. The manipulations with complexes 3 and 4 did not require an inert atmosphere. Solvents were purchased from Acros Organics (Geel, Belgium). The solvents used were dried and purified by distillation under inert atmosphere. The ESI_pos mass spectra were recorded with an AmazonX (Bruker Daltonics GmbH, Bremen, Germany) spectrometer at a capillary voltage of 4500 V. The mass spectrometry data were solved using the DataAnalysis 4.0 (Bruker Daltonics GmbH, Bremen, Germany) program. The mass spectra are presented as m/z values. The ¹H NMR (400 MHz and 600 MHz) and ³¹P NMR (162 and 242 MHz) spectra were recorded using Bruker Avance-DRX 400 and Bruker Avance-600 spectrometers (BrukerBioSpin, Billerica, MA, USA). The chemical shifts (δ) and coupling constants (J) are reported in ppm and in Hz, respectively. The internal standard for ¹H NMR is SiMe₄, and the external standard for ³¹P NMR is 85% H₃PO₄ (aq). A CHN analyzer “CHN-3 KBA” was used for the determination of the CHN content. The determination of the phosphorus content was provided by combustion in an oxygen stream. The starting pyridylphosphines were prepared according to procedures in the literature.