Hplc grade

The carbohydrate content was measured utilizing high-performance liquid chromatography (HPLC) with a Sykam instrument (Fürstenfeldbruck, Germany). Sample preparation and chromatographic analysis were performed following the procedures described in the study by Ziarno et al. (2019) . Briefly, 8 g of each sample was homogenized with 32 g of methanol (HPLC grade; Sigma-Aldrich, St. Louis, Missouri, United States) using an automatic shaker and an ultrasonic bath (for 30 min). The samples were then centrifuged (16,000× g, 4°C, 30 min) and filtered through syringe filter (0.22 μm). The analysis was carried out with a guard column Sugar-D (10 mm × 4.6 mm, 5 μm; Cosmosil, Nacalai Tesque, Kyoto, Japan) and column Sugar-D (250 mm × 4.6 mm, 5 μm; Cosmosil). The chromatographic separation parameters were as follows: flow 1 mL/min, oven temperature 30°C, range of detector 10,000 mV, and sample rate 2 Hz. The mobile phase was a 60: 40 mixture of acetonitrile (HPLC-grade, Sigma-Aldrich) and deionized water. Carbohydrate concentration was calculated based on the standard curves (of fructose, galactose, glucose, sucrose, raffinose, stachyose, and verbascose; HPLC grade; Sigma-Aldrich, St. Louis, Missouri, United States) and expressed as milligrams of sugar per 100 g samples (mg%). The presented data represents the average values obtained from five independent replications.

A volume of 100 μL plasma was mixed with 100 μL H₂O (HPLC grade, Merck Millipore, USA), and 600 μL methanol (HPLC grade, Merck Millipore, USA) was added. Samples were vortexed and incubated overnight at −20°C for protein precipitation. After centrifugation (13.000 × g, 15 min, 4°C), the supernatant was removed, dried using a SpeedVAC concentrator (SVC100H, SAVANT, Thermo Fisher Scientific, Illkirch, France), resuspended in 80 μL 20:80 acetonitrile-H₂O mixture (HPLC grade, Merck Millipore) and stored at −20°C until use for metabolomic analyses.

For extraction of quercetin, plasma (200 µL) was mixed with 400 µL methanol (99.9%, HPLC grade, Merck, Germany) and HCl (200 µL, 25%, analytical grade, Merck, Germany). The resulting mixture was vortexed in a vortex shaker for 90 s and incubated at 50 °C for fifteen minutes [22 (link)]. Then, this mixture was centrifuged at 10,000 rpm for ten minutes. The supernatant (20 µL) was injected into the column for determination of quercetin in plasma. The mixture of Na₂HPO₄ (≥99%, HPLC grade, Merck, Germany) (30 mM), acetonitrile (99.9%, HPLC grade, Merck, Germany), and methanol (65:29:6, v/v/v) was used as a mobile phase at the flow rate of 1 mL min⁻¹. The column effluent was analyzed at 370 nm using a UV–Visible detector. The standard plots of quercetin were constructed, and pharmacokinetic parameters were determined using MS Excel, 2010. Maximum peak concentration in plasma (C_max), the time required for maximum concentration (T_max), was determined from concentration time curves. Area under curve (AUC) was determined using the trapezoidal method. Results were expressed as Mean ± SD (n = 3).