Waters oasis hlb

All reagents used are HPLC grade. Ultrapure deionized water, with resistivity of 18.2 megaohm-cm was obtained from a Milli-Q^® Integral water purification system with Q-pod (Millipore, Bedford, MA, USA). Formic acid was purchased from Carlo Erba (Milan, Italy). Ethanol, acetonitrile and mEthanol from Merck (Darmstadt, Germany). Ammonium acetate, acetone and 2-propanol were purchased from Carlo Erba (Milan, Italy). The SPE cartridges Waters Oasis HLB (200 mg) from Waters S.p.A. (Milan, Italy). Standard solutions of gallic acid, ascorbic acid, vanillic acid, caffeic acid, chlorogenic acid, catechin, epicatechin, vanillic acid, coumaric acid and hydroxybenzoic acid were purchased from Extrasynthese (Genay Cedex, France); quercetin, rutin, apigenin, luteolin, ferulic acid, kaempferol, palmitic acid, stearic acid, oleic acid, linoleic acid, glucose, N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA), trimethylchlorosilane (TMCS) and pyridine were acquired from Sigma-Aldrich S.r.l. (Milan, Italy).

A subgroup of seventeen subjects performed an additional bioavailability study, to
identify circulating phenolic metabolites following the administration of experimental
beverages. Halfway through the supplementation period (30 (sem 3) d), fasted
subjects were administered their respective treatment at INAF (SCP, n 8;
Control, n 9). Blood samples were collected using EDTA-containing
syringes before and 30, 60, 120, 240 and 360 min after the ingestion. During the
experiment, all subjects were kept fasted. Plasma samples were obtained by centrifugation
(3500 rpm, 10 min at 4°C). Plasma phenolic compounds were characterised by UHPLC–MS/MS as
previously described⁽²⁴⁾, with slight modifications. Acidified plasma samples (300 µl) were loaded
into preconditioned Waters OASIS HLB (Waters Ltd) µElution plates 2 mg–30 µm. The retained
phenolic compounds were eluted with 75 µl of acetone–ultrapure water–acetic acid solution
(70:29·5:0·5, v/v/v) in presence of rosmarinic acid as internal standard (1 µg/ml final
concentration). The eluted solutions were directly analysed by UHPLC–MS/MS, using a Waters
Xevo TQD MS (Waters Ltd) coupled to a Waters Acquity UHPLC (Waters Ltd). Phenolic
metabolites were separated and identified as previously reported⁽²⁴⁾.

Organic acids and sugar were analyzed with HPLC on a 1,260 Infinity Agilent HPLC system. Approximately 100 mg of N₂-ground sample powder was diluted in 1 mL of deionized water and homogenized. Then 500 µL of sample solution was added to 4.5 mL of 0.65 mM sulfuric acid solution (H₂So₄), pre-treated by solid phase extraction with Waters Oasis HLB, 6 cm³ (200 mg) cartridges (Waters Corporation, Milford, MA, USA) and subsequently filtered through 0.2 µm nylon filters (Millipore, Burlington, MA, USA). A volume of 20 μL per sample was injected onto an Aminex HPX-87H column 300 × 7.8 mm (Bio-Rad Laboratories, Hercules, CA, USA) and eluted under isocratic conditions at 80 °C with 0.65 mM H₂SO₄ solution mobile phase at a 0.5 mL min⁻¹ flow rate. Eluting organic acids were detected with UV absorbance (210 nm), and the refractive index was measured with a Waters 2487 dual absorbance detector (Waters Corporation, Milford, MA, USA). A Kontron 475 refractive index detector (Kontron Instruments, Rossdorf, Germany) was used to determine fructose and glucose concentrations. Concentrations were calculated according to Eyéghé-Bickong et al.⁵² .