Kinetex c18 column

Lipid mediators in human plasma were analysed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) based on protocols published previously [12] (link), [13] (link). Briefly, samples were collected and stored immediately at −80°C. Plasma samples (500 µL) were defrosted on ice and adjusted to 15% (v/v) methanol: water (final volume 4 mL). Internal standards, PGB2-d4 (40 ng) and 12-HETE-d8 (40 ng) (Cayman Chemical Company, Ann Arbor, USA) were added and the pH of resulting solutions adjusted to 3.0 (1 M HCL). Acidified samples were immediately applied to preconditioned solid-phase cartridges (C18-E, Phenomenex, Macclesfield, UK) and lipid mediators eluted with methyl formate. LC/ESI-MS/MS analysis was performed on a HPLC pump (Waters Alliance 2695) coupled to an electrospray ionisation triple quadrupole mass spectrometer (Quattro Ultima, Waters, UK). Chromatographic separation was performed on a C18 Luna column (5 µm, 150×2.0 mm, 21 Phenomenex) for eicosanoids and a C18 Kinetex column (2.6 µm, 100×2.1 mm, Phenomenex) for hydroxy-fatty acids. Analytes were monitored on multiple reaction monitoring mode as reported [12] (link), [13] (link) with the following additions: 15-hydroxyeicosatrienoic acid (HETrE) m/z 321>221, 10-hydroxydocosahexaenoicacid (HDHA) m/z 343>153, 14-HDHA m/z 343>161, 13-HDHA m/z 343>193 and 17- HDHA m/z 343>201.

High-performance liquid chromatography was performed with the Jasco LC Net II, equipped with the AS-4150 autosampler, the PU-4180 pump and the MD-4010 PDA detector. The system was controlled with the JASCO ChromNAV Version 2.01.00 (JASCO International Co., Ltd., Tokyo, Japan). The experiments were performed on a C18 Kinetex column (150 mm × 4.6 mm, 2.6 μm; Phenomenex, Torrance, CA, USA). The mobile phase consists of A (water containing 1% formic acid) to B (methanol containing 1% formic acid). The gradient program was from 95% A to 80% in 10 min, from 80% to 70% in 5 min, from 70% to 50% in 5 min, from 50% to 0% in 5 min and isocratic for 10 min at a flow rate of 1 mL/min. Five µL of the sample was injected in duplicate onto the column kept at 50 °C.
The UV-Vis absorption spectra of the standards, as well as the samples, were recorded in the range of 190 to 600 nm. Polyphenols were detected at 280, 320 and 360 nm (Figure 3) and identified by the comparison of their retention times and UV-Vis spectra to those of pure standards. Quantification was performed by external standard calibration. The amount of polyphenols was expressed as mg/100 g of dw.

The concentration of FXII900 in the plasma samples was quantified based on peak intensities of total ion current (TIC) chromatograms acquired by LC-MS (LCMS-2020, Shimadzu). To 15 μl of plasma sample, 1 μM of internal standard peptide and 5 μl of 6 M guanidinium hydrochloride solution were added and mixed. Plasma proteins were precipitated by the addition of 400 µl of ice cold ethanol (99.9% [v/v] EtOH, 0.1% [v/v] TFA) and incubated on ice for one hour. Precipitate was removed by centrifugation (9000 × g, 20 min, 4 °C) and the supernatant dried by centrifugal evaporation under vacuum. Dried samples were dissolved by sequentially adding 2 μl of DMSO and 18 μl of H₂O containing 0.1% (v/v) CHOOH and analyzed by LC-MS. The samples were analyzed using an analytical C18 column (Phenomenex C18 Kinetex column, 50 × 2.1 mm, 2.6 µm, 100 Å) and a linear gradient of 5–30% solvent B (MeCN, 0.05% [v/v] CHOOH) in solvent A (H₂O, 0.05% [v/v] CHOOH) in 4.5 min at a flow of 1 ml per min. The mass was measured on a single quadrupole mass spectrometer in positive ion mode using electrospray ionization. Peptides were quantified based on the absolute intensities of the detected mass peaks (M³⁺ and M⁴⁺).

Mycelium and the agar directly underneath the membrane were harvested into glass scintillation vials as separate samples from each plate after 5 days of incubation in the dark at 30°, in six replicates, along with media controls (PDA with sterile membrane overlaid) and immediately frozen at −20°. Thawed samples were extracted by immersion in ethyl acetate (gently shaking for 1.5 h), dried under vacuum, and then reconstituted in MeOH to a concentration of 500 µg/ml. The ultra-high performance liquid chromatography coupled high resolution mass spectrometry (UPLC-HRMS) analysis was carried out on a Thermo Ultimate 3000 UPLC coupled to a Thermo LTQ Orbitrap XL high-resolution mass spectrometer, using a reverse-phase Phenomenex C18 Kinetex column in ESI+ mode (with an m/z 100–2000 m/z range). Data preprocessing closely followed the methodology of Overy et al. (2017) , using MZMine v2.29 [Cell Unit, Okinawa Institute of Science and Technology (OIST), Onna, Okinawa, Japan], with a mass detection noise cutoff level set to $5.0\times {10}^5$ . Mass features (each representing an associated retention time and mass/charge ratio) from agar and mycelium extracts were normalized to the total ion current detected from each sample and then summed, scaled, and centered.

NODAGA-E[c(RGDyK)]₂ acetate was obtained from ABX GmbH (Radeberg, Germany). Gallium-68 (t_1/2 = 68 min; E_max,_β+ = 1.90 MeV (89%)) labelling of NODAGA-E[c(RGDyK)]₂ acetate was performed using a Modular-Lab eazy module (Eckert & Ziegler, Berlin, Germany). The ⁶⁸Ge/⁶⁸Ga generator (IGG100, Eckert & Ziegler) was eluted with 6 mL 0.1 M HCl. The eluate was concentrated on a Bond Elut SCX cartridge and eluted with 600 µL 5 M NaCl/5.5 M HCl (41:1). NODAGA-E[c(RGDyK)]₂ (30 nmol) was labelled in 1000 µL 0.7 M NaOAc buffer pH 4.5 and 400 µL 50% EtOH at 60 °C for 400 s. The resulting ⁶⁸Ga-NODAGA-E[c(RGDyK)]₂ was formulated with saline or phosphate buffer.
The radiochemical purity was more than 96% on HPLC, and the amount of unlabeled ⁶⁸Ga in the product was less than 1%, as demonstrated by radio–thin layer chromatography.
All reagents and cassettes were purchased from Eckert & Ziegler. For analysis, a high-performance liquid chromatograph (Ultimate 3000; Dionex, Sunnyvale, CA, USA) was used with a 2.6-μm, 100-Å, 50 × 4.6 mm C18 Kinetex column (Phenomenex, Torrance, CA, USA). The mobile phases were: eluent A: 10% MeCN in H₂O with 0.1% trifluoroacetic acid; eluent B: 10% H₂O in MeCN with 0.1% trifluoroacetic acid.