Suprapur grade nitric acid

High-purity deionized water acquired by passing distilled water through a water purification system (demineralizer HLP 20, Hydrolab, Poland) was used to prepare the solutions. Certified single-element standard solutions (1000 mg/L) used to prepare the calibration curve were of the highest purity grade (99.999%) and were supplied by Ultra Scientific (North Kingstown, RI, USA). Other reagents were of analytical grade unless otherwise stated. Honey samples were digested with Suprapur® grade nitric acid (HNO₃ 65% m/m, Merck, Germany). A recovery test was performed using a single-element solution and two reference materials, namely, Standard Reference Materials (SRMs) Tomato Leaves and Pine Needles (Pinus taeda) (SRM 1573a and SRM 1575a, resp., National Institute of Standards and Technology (NIST), Gaithersburg, USA).

The non-heme hepatic and placental iron content was determined by acid digestion of the samples at 100 °C for 10 min, followed by colorimetric measurement of an iron-ferrozine complex (absorbance at 560 nm, Beckman DU-68) as described previously [14 ].
A 20 µL sample of colostrum was diluted in 2 ml of boiling Suprapur-grade nitric acid (Merck, Darmstadt, Germany). The total iron concentration was then measured using the graphite furnace atomic absorption spectrophotometry (AAS) technique (AAnalyst 800, Perkin-Elmer, Waltham, MA, USA). Three samples of a standard reference material (197.94 ± 0.65 Fe mg/kg), were analyzed for normalization of the obtained data.

The copper content in the kidney and liver samples obtained from the experimental male mice was measured by atomic absorption spectrophotometry. The samples were weighed and digested in 2 mL of boiling Suprapur-grade nitric acid (Merck, Darmstadt, Germany). After cooling to room temperature (RT), each sample was suspended in 10 mL of deionized water. The reference material samples were prepared in a similar manner. The copper concentration was measured using the graphite furnace AAS technique (AAnalyst 800, Perkin-Elmer, Perkin-Elmer, Waltham, MA, USA). Three samples of nitric acid were used as blanks. In addition, three samples of standard reference material, Cu = 189 ± 4 mg/kg⁻¹, were analyzed for the normalization of the obtained data.