Dionex ics 6000 hpic system

The HPIC separation was carried out using a Thermo Scientific Dionex ICS-6000 HPIC System (Thermo Scientific) equipped with Dionex IonPac AS11-HC (2 × 250 mm) and AG11-HC (2 mm × 50 mm) columns. Mobile phase A was 100 mM NaOH in water, and phase C was water. Another pumping system was used to supply the solvent (2 mM acetic acid in methanol) and the solvent was mixed with effluent before entering the ion source (flow rate of 0.15 mL/min). The column temperature was set at 30 °C. The auto-sampler temperature was set at 4 °C, and the injection volume was 5 μL.
A QE mass spectrometer was used for its ability to acquire MS spectra in full ms mode via the acquisition software (Xcalibur 4.0.27, Thermo). In this mode, the acquisition software continuously evaluated the full scan MS spectrum. The ESI source conditions were set as follows: sheath gas flow rate as 30 Arb, Aux gas flow rate as 10 Arb, capillary temperature 350 °C, full MS resolution as 35,000, and spray Voltage as − 3.8 kV (negative).
The raw data were converted to the mzXML format using ProteoWizard (Massconvert) and were processed for peak detection, extraction, alignment, and integration using an in-house program that was developed using R and XCMS.

Tissue samples were added to 500 µL of precooled MeOH/H₂O (3/1, v/v). They were then homogenized for 4 min at 40 Hz and sonicated for 10 min in an ice water bath. Homogenization and sonication cycles were repeated three times, followed by incubation at −40 °C for 1 h and centrifugation at 12000 rpm and 4 °C for 15 min. The supernatants (400 µL) were collected and dried. Then, 250 µL of water was added to the dried residue to create a reconstituted solution. The reconstituted samples were vortexed before filtration through a filter membrane, and subsequently transferred to inserts in injection vials for HPIC‐MS/MS analysis. HPIC separation was performed using a Thermo Scientific Dionex ICS‐6000 HPIC System (Thermo Scientific) equipped with Dionex IonPac AS11‐HC (2× 250 mm) and AG11‐HC (2 mm×50 mm) columns. An AB SCIEX 6500 QTRAP⁺ triple quadrupole mass spectrometer (AB Sciex) equipped with an electrospray ionization (ESI) interface was used for assay development. The typical ion source parameters were IonSpray voltage = ‐4500 V, temperature = 450 °C, ion source gas 1 = 45 psi, ion source gas 2 = 45 psi, and curtain gas = 30 psi. AB SCIEX Analyst Work Station software (1.6.3 AB SCIEX), MultiQuant 3.0.3 software, and Chromeleon7 were employed for MRM data acquisition and processing.

High-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) was performed with a Dionex ICS-6000 HPIC system (Thermo Fisher Scientific) for quantification of α-1,4-glucans and acarbose. Carbohydrates were separated at 35 °C with a Dionex CarboPac PA100 column (250 × 4 mm, 8.5 µm, Thermo Fisher Scientific) coupled with a Dionex CarboPac PA100 Guard column (250 × 4 mm, 8.5 µm, Thermo Fisher Scientific). Pulsed amperometric detection was performed with the gold, carbo, quad waveform using a non-disposable gold electrode and an AgCl reference electrode at a system temperature of 30 °C. Flow rate was set at 1 mL min⁻¹. An amount of 20 µL of the diluted sample (40- and 400-fold) was injected to the system. Eluent A (166 mM ammonium hydroxide) and eluent B (1 M sodium acetate with 166 mM ammonium hydroxide) were applied using the following gradient: 6.5 min 10% B, 31.5 min 25% B, 34.0 min 25% B, 44.0 min 10% B. Evaluation of the data was executed with Chromeleon Chromatography Data System 7.2.10 software (Thermo Fisher Scientific).