Ed50 electrochemical detector

Fructans concentration was measured by gel permeation chromatography (GPC) using a HPLC equipped with a linear Ultrahydrogel column (Waters, Japan) using running conditions described by Porras-Dominguez et al. (16 (link)), 0.8 mL/min of 0.1 mM NaNO₃ at 30°C as eluent. Fructans profile was determined by HPAEC-PAD, using an ED50 electrochemical detector (Dionex, USA) and a CarboPac® PA200 column for carbohydrate analysis (Thermo scientific, USA) using running conditions stablished by Mellado-Mojica and López (17 (link)). Product elution was carried out by applying a sodium acetate gradient with 100 mM NaOH at 0.5 mL min⁻¹ as follows: 5–100 mM sodium acetate in 25 min, 100–400 mM in 60 min, and 10 min for initial condition re-equilibration (5 mM sodium acetate) at 30°C. We determined the number average molar mass (Mn), mass average molar mass (Mw), and polydispersity index (PI) for each sample, as described by Porras-Dominguez et al. (16 (link)). From these data we estimated the degree of polymerization by number (DPn) and the degree of polymerization by mass (DPw) using linear regression analyses with data for glucose, sucrose, 1-ketose, nystose, fructosyl-nystose, as well as 5.2, 11.6, 23.8, and 48.6 KDa dextran as standards.

For the determination of mannitol content, mycelia were freeze-dried and ground into powder by a TissueLyser (Qiagen, Hilden, Germany). The method for determining mannitol levels in V. volvacea mycelia by high-performance anion chromatography-pulsed amperometric detection (HAPEC-PAD) has already been described [20 , 21 (link)].
The samples (0.1 g) were ultrasonically extracted in 10 mL of ultrapure water for 30 min and centrifuged at 3600 × g for 20 min at temperature of less than 25°C. Then, the supernatant was filtered with a 0.45-μm filter (Millipore, Bedford, MA, USA) and diluted 5 times for analysis by an ICS2500 HPAEC-PAD system comprising a GP50 quaternary gradient pump, an EG50 automatic eluent generator, an LC30 column oven, a Dionex CarboPac MA1 column, and an ED50 electrochemical detector (Dionex, Sunnyvale, CA, USA). The temperature of the column was 30°C, the mobile phase was 480 mM NaOH solution, and the flow rate was 6.67 μL s⁻¹. The adopted external standard mixture included mannitol (Sigma, USA). Each standard was dissolved in deionized water and diluted to several standard solutions to generate a calibration curve [22 (link)].

Quantification of sialyllactose was carried out by HPAEC-PAD analysis using a Dionex BioLC system consisting of GS50 gradient pumps, ED50 electrochemical detector, AS50 chromatography compartment coupled to an AS50 autosampler (Dionex Corp., Sunnyvale, CA). Samples (10 µL) were injected on a CarboPac™ PA1 (4 mm×250 mm) analytical column (Dionex Corp., Sunnyvale, CA) at a flow rate of 1 mL/min. The elution program was based on the method described in [20] except for the modifications in the eluent system given below. The eluent system comprised of deionised water (A), 0.5 M NaOH (B), 1 M NaOAc (C). For the first 3 min an isocratic elution of 80: 20 (% A:B) was applied, which was followed by a linear gradient from 80∶20 (% A:B) to 60∶20∶20 (% A:B:C) from 3 to 27 min. Strongly retained anions were removed from the column by isocratic elution at 40∶20∶40 (% A:B:C) from 27 to 31 min. Subsequently, the column was re-equilibrated for 7 min with 80∶20 (%A:B).

The analysis of the hydrolyzed samples was performed with a High-Performance Anion Exchange Liquid Chromatography with Pulsed Amperometric Detector (HPAEC-PAD) Dionex system composed of LC25 Chromatography Oven, GP50 Gradient Pump, AD25 Absorbance Detector, ED50 Electrochemical Detector, and equipped with a Column CarboPac PA10. The flow rate was maintained at 1 mL/min at room temperature. The injection volume was 20 mL. HPLC-grade H₂O was used as eluent A, while 0.05 and 0.8 M NaOH were used as eluent B and C, respectively. The gradient system was as follows: 0–20 min, 60% A and 40% B; 20–27 min, 75% A and 25% C; 27–31 min, 100% C; 31–42 min, 60% A and 40% B. Standard solutions of fucose, galactosamine, glucosamine, galactose, glucose, and mannose were prepared and analyzed at different concentrations in order to obtain a calibration curve for each analyte. Limit of detection (LOD) and limit of quantification (LOQ) were 0.003 and 0.009 µg/mL, respectively. Each sample was analyzed in triplicate and quantified by interpolation of the area of the peak. Chromeleon Client software v 6.80 was used to process the raw chromatograms. A data table with the concentrations of the analytes for each sample was finally obtained.