4.1 Chemicals The magnetic particles Dynabeads® MyOneTM Carboxylic Acid were from Invitrogen (Life Technologies, Van Alley, CA, USA). The hepcidin standard was obtained from Peptide Institute Inc (Osaka, Japan), N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 2-(N-morpholino)-ethane sulfonic acid (MES) from Calbiochem® (Merck), and phosphate buffer saline (PBS) from Sigma Aldrich. The solvents used, such as formic acid and acetonitrile, were LC-MS grade and were purchased from Panreac (Castelar del Vallés, Barcelona, Spain). The ultra-high quality (UHQ) water was produced using a Millipore water purification system.

4.2 Instrumental - UHPLC-MS/MS analysis Chromatographic analyses of hepcidin levels in plasma …show more content…
Hepcidin solution (20 μL) was added to the plasma before extraction with the beads, whilst in parallel another sample of plasma underwent extraction and then the same amount of hepcidin was added. The percentage of recovery was calculated as (AreaPRE / AreaPOST) x 100, where AreaPRE represents the samples with hepcidin added before bead treatment and AreaPOST the samples with hepcidin added after bead treatment. The values obtained were above 90 % in all cases.

4.4. Dietary intervention The functional juice aronia-citrus juice (ACJ) included in this study was a mixture of citrus juice (95%) and 5% aronia extract (Aronia melanocarpa), based on a drink model developed before and reported by Gonzalez-Molina et al [36]. The juice was developed on an industrial pilot scale by a company of Murcia region [27]. The nutritional composition of the ACJ was: energy 38 kcal/100 mL; protein 0.45 g/100 mL; carbohydrates 9 g/100 mL; fat 0.03 g/100 mL. Vitamin C content of the juice was 30 mg per 100 mL. The contents of fruit flavonoids, hydroxycinnamates, and catechins are summarized in Table 21