Suprapur nitric acid

Suprapur^® nitric acid (65%) and Suprapur^® hydrogen peroxide (30%) were purchased from Merck (Darmstadt, Germany). Quadruple-distilled water with a conductivity of less than 1 µS/cm was obtained using an S2–97A2 distillation apparatus (Chemland, Stargard Szczecinski, Poland). Standards of Zn(II), Fe(III), Mn(II), Mg(II), and Cu(II) at concentrations of 1 g/L were purchased from the District Office of Measures in Łodz, Poland.
The reagents used for the analysis of phenolic acids in the samples were as follows: MeOH and glacial acetic acid of analytical grade were purchased from Chempur, and MeOH of HPLC grade was purchased from Merck. The following standards were purchased: caffeic acid, chlorogenic acid, neochlorogenic acid, and rutoside (Sigma Aldrich, St. Louis, MI, USA); cryptochlorogenic acid, isochlorogenic acid, 4-feruloylquinic acid, and astragalin (ChromaDex, Los Angeles, CA, USA); and caffeine (Fluka Chemie AG, Buchs, Switzerland).

Reagents for antioxidant activities and cholinesterase inhibition assays were from Sigma-Aldrich (St. Louis, MO, USA and Steinheim, Germany), except magnesium chloride hexahydrate, which was bought from VWR (Leuven, Belgium), sodium chloride from Fisher Scientific (Fair Lawn, NJ, USA), sodium nitroprusside dihydrate from Fluka, and sodium carbonate from Merck. Hydrogen peroxide 35.5% w/w was purchased from Labchem (Zelienople, PA, USA). Methanol Chromasolv for HPLC was from Riedel-de Haën (Seelze, Germany), and formic acid was from Carlo Erba (Val de Reuil, France). The standards used for HPLC analyses were acquired from Sigma-Aldrich (4-O-caffeoylquinic acid, quercetin-3-O-rutinoside hydrate, protocatechuic acid, ferulic acid, and p-coumaric acid), Fluka (caffeic acid), Alfa Aesar (5-O-caffeoylquinic acid), and Extrasynthèse (Genay, France): 3-O-caffeoylquinic acid, apigenin-8-C-glucoside, apigenin-6-C-glucoside, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, kaempferol-3-O-rutinoside, and tiliroside. Ultrapure water (with resistivities of 18.2 MΩ/cm) was obtained using a Milli-Q water purification system from Millipore (Molsheim, France). Standard solutions of Pb and Cd, Atomic Absorption standard with 4% HNO₃, were purchased from SPC Science (Quebec, Canada). Suprapur nitric acid 65% (v/v) and NH₄H₂PO₄ (Suprapur) were from Merck (Darmstad, Germany).

Molybdenum content in purified protein samples was quantified with an Agilent 7700 Series ICP-MS (Agilent Technologies) using a standard calibration curve of inorganic molybdenum (Fluka). The molybdenum calibration for the inductively coupled plasma mass spectrometry (ICP-MS) was in the range of 0.1–20 µg/l. Samples were mixed with rhodium (Rh(NO₃)₃) as an internal standard. Gathered data were processed using MassHunter work station software. Protein samples with concentrations of 0.8–1.5 mmol/l were used for analysis. Samples were diluted 1:1 in 69% Suprapur^® nitric acid (Merck), after an overnight incubation at room temperature they were diluted with Milli-Q water prior to ICP-MS measurements.

The serum samples were defrosted; 200 µL of serum was transferred to digestion vessels (DigiTUBE SCP SCIENCE 50 mL class A) and mixed with 1.5 mL of 65% Suprapur^® nitric acid (Merck, Darmstadt, Germany) and 5.0 mL of deionized water. Then, vessels were placed in heating blocks (DigiPREP SCP SCIENCE) and digested for 60 min at 120 °C. After digestion, vessels with solution were left to reach room temperature (RT) and were filled with deionized water to 10 mL. The analysis was performed using PlasmaQuant PQ 9000 Analytik Jena AG. The following operating conditions of ICP-OES were used: power 13,000 W, plasma gas 14.0 L/min, auxiliary gas 0.50 L/min, nebulizer gas 0.60 L/min, and the monitoring direction of the plasma flame was axial. Standard solution for calibration curves of magnesium at the concentration of 1000 µg/L was prepared by diluting magnesium 1000 mg/L standard (PlasmaCAL SCP SCIENCE) with 0.5% nitric acid in deionized water. The analysis line used for magnesium quantification was 279.08 nm. Each serum sample was run in triplicate.