Vanquish lc system

5 µl of each sample or the glucose and fructose standard mixture in 90% acetonitrile was injected into a Vanquish LC system (ThermoFisher Scientific, UK) using a flow rate of 0.25 mL min⁻¹. The analytical column was Accucore-150-Amide-HILIC (2.6 µm particle size, 150 mm × 2.1 mm, ThermoFisher Scientific, UK) held at 35 °C. Starting mobile phase composition was 90% solvent B (0.1% ammonium acetate in acetonitrile) in A (0.1% ammonium acetate and water) decreasing to 60% B over 5 minutes and held constant for 4 minutes before being re-equilibrated to 90% B after 2 minutes. Column eluant was eluted into an Orbitrap Exploris 240 mass spectrometer (ThermoFisher Scientific, UK) and ionised using electrospray ionisation in negative polarity at 2500 V. Mass measurement used full scan mode resolution of 120,000, a m/z range of 5–500. The maximum injection time was set automatically by the software. Samples and standards were analysed in triplicate and Tracefinder 5.1 (ThermoFisher Scientific, UK) was used to construct the calibration curve and determine peak areas of hexose signals in the samples. Calibration standard concentrations analysed were 1 ng, 10 ng, 100 ng, 1 µg and 10 µg. Final data were normalised per cm of hyphae per g of starting material.

Analysis was conducted using a Vanquish LC system coupled to a Q‐Exactive Focus Orbitrap mass spectrometer (Thermo, Hemel Hempstead, UK). The LC was equipped with a Waters X‐Select HSS T3 C18 column (2.1 × 75 mm, 2.5 μm) using 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B) as mobile phases. A gradient was utilised, with flow rate 0.48 ml/min, starting at 0% B for 1 min increasing to 10% B within 2.2 min, 60% within 4 min and increasing to 100% B within 6.5 min. The system was held at 100% B for 1 min prior to re‐equilibration at starting conditions.
The mass spectrometer was operated in positive ionisation mode with a HESI‐II (Heated Electro Spray Ionisation) probe using a vaporiser temperature of 420°C and ion transfer tube temperature of 320°C. The spray voltage was 2500 V. The mass spectrometer was operated in full scan data dependent Discovery (ddDiscMS/MS) mode to capture both MS and MS² data concurrently. MS² data were derived from the fragmentation of ions detected above a threshold. Subsequent analysis was performed utilising Parallel Reaction Monitoring (PRM) mode to ensure MS² data were captured for both higenamine and the IS. Data were processed using Thermo XCalibur version 4.0.27.21 QualBrowser and Thermo TraceFinder version 4.1.

Single reaction monitoring mode was used to detect glycocholic acid on a Altis QQQ Mass Spectrometer equipped with a Vanquish LC system (Thermo Fisher Scientific). Separation of metabolites was performed on a Acquity HSS T3 column (Waters; 150 mm by 2.1 mm, 1.8 μm). The mobile phase consisted of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid) using the following gradient: 0 min 20% B, 8 min 95% B and 10 min 20% B, at a constant flow rate of 0.3 ml min⁻¹. The injection volume was 5 µl. Two transitions were optimized using an authentic standard of glycocholic acid (glycine-1-¹³C, CLM-191-PK, Cambridge Isotope Laboratories), from the negative precursor ion (m/z 465) to product ions (m/z 402 and m/z 75). Total cycle time was 0.8 s and Q1 resolution (FWHM) was 0.7 and Q3 resolution (FWHM) was 1.2. For each transition, the collision energy applied was optimized to generate the greatest possible signal intensity. The optimized source parameters were: spray voltage, 2,500 V; sheath gas, 35; Aux gas, 7; ion transfer tube temperature, 325 °C; vaporizer temperature, 275 °C; and RF lens, 105. Data acquisition was performed using Xcalibur 4.1 software.

For LC-MS analysis, dried samples were re-dissolved in 300 μl acetonitrile:water (80:20; v/v). All measurements were carried out on a Q Exactive HF mass spectrometer coupled with a Vanquish LC system (Thermo Scientific). A sample volume of 5 μl were separated on a Waters Acquity UPLC CSH C₁₈ column (100 × 2.1 mm; 1.7 μm) coupled to an Acquity UPLC CSH C₁₈ VanGuard precolumn (5 × 2.1 mm; 1.7 μm). The column was maintained at 65 °C at a flow rate of 0.6 ml min^− 1. The mobile phases consisted of A: acetonitrile:water (60:40, v/v) with ammonium formate (10 mM) and formic acid (0.1%) and B: 2-propanol:acetonitrile (90:10, v/v) with ammonium formate (10 mM) and formic acid (0.1%). The 15 min separation was conducted under the following gradient: 0 min 15% B; 0–2 min 30% B; 2–2.5 min 48% B;2.5–11 min 82% B; 11–11.5 min 99% B; 11.5–12 min 99% B; 12–12.1 min 15% B; 12.1–15 min 15% B. Orbitrap MS instrument was operated in electrospray ionization (ESI) in positive mode with the following parameters: mass range 60–900 m/z; spray voltage 3.6 kV, sheath gas (nitrogen) flow rate 60 units; auxiliary gas (nitrogen) flow rate 25 units, capillary temperature 320 °C, full scan MS1 mass resolving power 120,000, data-dependent MSMS (dd-MSMS) 4 scans per cycle, dd-MSMS mass resolving power 30,000. Thermo Xcalibur 4.0.27.19 was used for data acquisition and analysis [73 (link)].

Heart sample extracts were reconstituted in a 10 mM ammonium acetate solution/internal standard mixture (200 µL; phenylalanine d5, valine d8, leucine d10), while coronary effluent samples were diluted (20 µl plus 80 µl 10 mM ammonium acetate/internal standard mix). All samples were run for 6 min using an ACE Excel-2 C18-PFP 5 μm column (100 A, 150 x 2.1 mm, 30°C) on either a Thermo Vanquish LC system coupled to a Thermo Quantiva triple quadrupole mass spectrometer for targeted analysis or a Thermo Dionex Ultimate 3000 LC system coupled to a Thermo Elite orbitrap mass spectrometer for open profiling. Mobile phase A was 0.1% formic acid, while mobile phase B was acetonitrile plus 0.1% formic acid. The LC gradient was as follows: 0% B for 1.6 min followed by a linear gradient up to 30% B for 2.4 min. There was a further linear increase to 90% B for 30 s, following which B was held at 90% for 30 s before re-equilibration for 1.5 min. Drying gas was as used in the BEH amide method.