6 n hcl

Quantitative and qualitative determination of phenolic compounds was analysed by HPLC method, as described by Hertog et al. [44 (link)] and modified by Remiszewski et al. [40 ]. For the determination of phenolic compounds, the extract obtained earlier was subjected to acid hydrolysis in the presence of 6 N HCl (Sigma-Aldrich, Munich, Germany); then, the mixture was incubated at 90 °C for 2 h. The samples prepared in this way were filtered through filters with a diameter of 0.45 µm (Millipore) and subjected to chromatographic analysis. Separation of the compound mixture was carried out on an X-Terra C18 RP column (150 × 3.9 mm i.d. 5 μm) from Waters at 30 °C. The injection volume was 10 µL, and the flow rate of the mobile phase was 1 mL/min. The eluted fractions were monitored by UV–VIS detector (Waters Alliance, Taunton, MA, USA). The measurement time was 50 min. Formic acid (>98%), methanol (99%), and water were used as mobile phase (Sigma-Aldrich, Munich, Germany). A linear concentration gradient from 5% to 60% methanol was applied with a constant concentration of 5% Formic acid. The recording of the chromatographic spectra was carried out in the wavelength range from 240–520 nm. Compounds were identified on the basis of spectra and retention times comparable to the standards (Sigma-Aldrich, Munich, Germany).

The macro elements (Ca, K, Mg) were detected using atomic absorption spectroscopy (AAS) after the acidic dissolution of the organic matrix. In this regard, 3 g of sample was dissolved in 6 N HCl (Sigma-Aldrich Chemie GmbH, München, Germany), and kept overnight in an acidic solution. After filtration, the solution was brought to a volume of 50 mL, with distilled water. The metal content was determined by atomic absorption spectrometry technique, using a Varian Spectra 240 FS spectrophotometer (Palo Alto, CA, USA), using the following protocol: an air:acetylene ratio of 13.50:2, nebuliser uptake rate of 5 L/min, and working solutions with concentrations ranging from 0.3 to 3 μg/L, prepared from multi-element ICP standard solution 1000 mg/L.
The samples were divided into equal parts to establish a relationship between the remineralisation protocols and the macro elemental composition. To better evaluate the mineral content in the region submitted to the different remineralisation protocols, we submerged the entire sample surface according to protocol, therefore, not having lesions smaller than the entire surface taken into analysis. Using AAS, the entire sample was analysed, and the data were used for statistical analysis. The results were expressed as the value obtained divided by the mass taken into analysis before acid immersion, so the values obtained were easily extrapolated.

Heme (Sigma-Aldrich, 52180) was prepared by adjusting the pH to 6.9–7.2 using 1N NaOH (Sigma-Aldrich) and 6N HCl (Sigma-Aldrich) solutions in PBS and then to a final concentration of 5 mg/mL in PBS. Mice were dosed at 50 mg/kg, i.p.

For total amino acid analysis, 10 mg of rice power of each sample was hydrolyzed with 1 mL of 6 N HCl (Sigma, USA) in a 2 mL screw-cap tube before adding 10 nmol l-(+)-norleucine (Wako Pure Chemicals, Japan). The samples were then heated at 110 °C for 24 h, followed by the treatment of 6 h at 65 °C in order to evaporate HCl completely. The residue was then dissolved in 1 mL Na-S™ buffer and centrifuged at 1600 × g for 10 min at room temperature. The supernatant was filtered with a 0.45 μm nylon membrane syringe filter (Pall Life Sciences, USA) and transferred to an autosampler bottle for amino acid analysis. HPLC data were normalized with the level of l-(+)-norleucine per sample. Three biological replicates were designed for each sample. Seventeen amino acids were measured, including alanine (Ala), arginine (Arg), aspartic acid (Asp), cysteine (Cys), glutamic acid + glutamine (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tyrosine (Tyr), and valine (Val).

The flag leaf blades at the heading stage and the mature seeds from transgenic plants, as well as WT, were harvested from the main tiller of each plant. For total amino acid analysis, 10mg of rice power of each sample was hydrolyzed with 1mL of 6N HCl (Sigma, United States) in a 2mL screw-cap tube before adding 10nmol L-(+)-norleucine (Wako Pure Chemicals, Japan). The samples were then heated at 110°C for 24h, followed by the treatment of 6h at 65°C in order to evaporate HCl completely. The residue was then dissolved in 1mL Na-S^TM buffer and centrifuged at 1,600 × g for 10min at room temperature. The supernatant was filtered with a 0.45μm nylon membrane syringe filter (Pall Life Sciences, United States) and transferred to an autosampler bottle for amino acid analysis. HPLC data were normalized with the level of L-(+)-norleucine per sample. Three biological replicates were designed for each sample. Seventeen amino acids were measured, including alanine (Ala), arginine (Arg), aspartic acid (Asp), cysteine (Cys), glutamic acid+glutamine (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tyrosine (Tyr), and valine (Val) (Yang et al., 2018 (link)).