Eurospher 2 100 5 c18

An isocratic high-performance liquid chromatography (HPLC) method was used to determine serum level of vitamin A (retinol) and E (α-tocopherol) simultaneously using UV detection [34 (link), 35 ]. Retinol acetate and tocopherol acetate were used as internal standards for retinol and α-tocopherol, respectively. The constituents of HPLC system consists are a LKB 2155 column oven, a LKB 2248 pump, and a 2140 rapid spectral detector (all of them are purchased from LKB, Pharmacia, Uppsala, Sweden). The isocratic mobile phase, methanol : acetonitrile : tetrahydrofuran (75 : 20 : 5), was pumped using a 5 μm reverse phase column (Eurospher II 100-5 C18, 250 × 4.6 mm) (Knauer, Berlin, Germany) at a flow rate of 1.5 mL/min. α-tocopherol and retinol are monitored at 285 nm and 325 nm, respectively.

Twenty μL of each extract (1 mg/mL) was separately injected into an analytical high-performance liquid chromatography system (HPLC) (Knauer, Berlin, Germany) by using an end capped Eurospher II 100-5 C18, Vertex Plus Column (Knauer, Berlin, Germany) (250 × 4.6 mm with precolumn) with particle size: 5 µm, pore size: 100 Å and temperature 30 °C; coupled to UV detector (Knauer GmbH-Smartline 2600, Berlin, Germany) at a wavelength range 190 to 500 nm (quantification at 330 nm), while MeOH/H₂O was applied as the mobile phase with a gradient system, increasing MeOH from 30% to 70% within 40 min, with a flow rate of 1 mL/min, at ambient temperature. Analysis was performed using SAS software, version 9.2 (SAS Institute Inc., Cary, NC, USA).

High performance liquid chromatography—HPLC - (DIONEX, Sunnyvale, CA, United States) was used to detect the sugar content and organic acids concentration. It was coupled with a refractive index detector (RI-71, SHODEX, Yokohama, Japan) and equipped with a Eurokat H column (300 mm × 8 mm × 10 μm; Knauer, Germany), eluted with 5 mM H₂SO₄ at 0.8 mL·min⁻¹. Cation analysis in the hydrolysate were performed by using an IonPac CS 16 column (250 mm × 4 μm, DIONEX, Sunnyvale, CA, United States), operating at flow rate of 1.0 mL·min⁻¹, at 40°C, with 30 mM CH₃SO₃H as mobile phase. Anion analysis was carried out utilizing IonPac AS9-HC column (250 mm × 4 μm, DIONEX; Sunnyvale, CA; United States), eluted with Na₂CO₃ at a flow rate of 1.2 mL·min⁻¹, at room temperature. Furfural, hydroxymethylfurfural and phenols were evaluated in a Dionex ICS 3000 system (Thermo Fisher Scientific) equipped with an Eurospher II 100-5 C18 with precolumn (Knauer, Germany) connected to a UV-visible detector (280 nm) using ultrapure water and 50% acetonitrile at 1 mL·min⁻¹. Ethanol was analysed using gas chromatography (GC) system (7890A, Agilent Technologies) equipped with DB-WAX Ultra Inert column (30 m × 0.25 mm length; d.f.: 0.25 µm) and a flame ionization detector (FID) at 300°C.