Iron standard solution

All reagents used were of reagent grade and, unless otherwise stated, were sourced from Sigma. Selectipur nitric acid was sourced from Lach:ner. Iron standard solution, a Certified Reference Material, was obtained from Merck (Darmstadt, Germany). Milli Q distilled deionised water was used throughout the experiments. All glass and polyethylene labware were soaked in concentrated nitric acid for 24 h and then rinsed with distilled deionised water before use to avoid contamination. Working solutions of enzymes were prepared immediately before use. Dietary supplements were purchased from the local pharmacy; the composition of the tablets is presented in the Table 1. Vitamins expressed as percentage of recommended daily intake in one tablet [21 ]. The weight of one tablet was 0.98 ± 0.04 g and 0.54 ± 0.02 g for dietary supplements 1 and 2 (Spl1 and Spl2), respectively.

To determine the total iron concentration in plant tissues, 0.1 g of sample was digested in 5 ml of the oxidizing mixture, i.e.,: 4.5 ml HNO₃ (69%) and 0.5 ml H₂O₂ (35%) at 180°C for 30 min using the closed microwave system (Milestone Ethos Plus, Sorisole, Italy). Mineralisation of soil samples (0.3 g) was performed with the use of the same mixture of 69% HNO₃ and 35% H₂O₂ (ratio 9:1) under the same conditions. Digested samples were transferred to plastic tubes and stored at 4°C. The amount of iron was measured by Flame Atomic Absorption Spectroscopy (FAAS) and Graphite Furnace Atomic Absorption Spectroscopy (GFAAS) using a Thermo Scientific SOLAAR M Series (TJA Solution, SOLAAR M, Cambridge, United Kingdom). In FAAS, the gas mixture was air and acetylene. The calibration curve range was 0–10 mg L⁻¹ and the lower limit of quantification was 0.01 mg L⁻¹. In GFAAS, graphite cuvette duty cycle was as follows: evaporation −100°C/30 s; incineration - 1,100°C/20 s; atomization - 2,100°C/3 s; cleaning - 2,500°C/3 s and the calibration curve range was 0–20 μg L⁻¹ with a lower limit of quantification of 0.1 μg L⁻¹. The deuterium lamp (TJA Solution, SOLAAR M, Cambridge, United Kingdom) was used for background correction. Iron standard solutions (Merck, Darmstadt, Germany) were prepared in 3% HNO₃.

The amount of iron was measured by Graphite Furnace Atomic Absorption Spectroscopy (GFAAS) using a Thermo Scientific SOLAAR M Series (TJA Solution, SOLAAR M, Cambridge, United Kingdom). The gas mixture was air and acetylene. The graphite cuvette duty cycle was as follows: evaporation—100 °C/30 s; incineration—1100 °C/20 s; atomization2,2-diphenyl-1-picrylhydrazyl 2100 °C/3 s; cleaning—2500 °C/3 s, and the calibration curve range was 0–20 µg/l with a lower limit of quantification of 0.1 µg/l. A deuterium lamp (TJA Solution) was used for background correction. Iron standard solutions (Merck, Darmstadt, Germany) were prepared in 3% HNO₃.