*4.9. Elemental Analysis*

Approximately 500 mg of each lyophilized pool were digested with 10 mL of HNO3, exploiting a closed-vessel microwave digestion system Ethos 1 (Milestone, Bergamo, Italy) equipped with PTFE vessels. The mineralization was carried out by setting the following temperature program; 0–200 ◦C in 10 min (step 1), 200 ◦C held for 5 min (step 2), and 200–220 ◦C in 5 min (step 3), with a constant microwave power of 1000 W. Due to the expected very high salt concentrations, each digested pool was diluted by ultrapure water with a dilution factor of 1000, and stored at 4 ◦C until ICP-MS analysis. A quadrupole ICP-MS iCAP Q (ThermoScientific, Waltham, MA, USA), equipped with an ASX-520 autosampler (Cetac Technologies Inc., Omaha, NE, USA), was employed for the analytical determinations. The ICP-MS operating conditions are shown in Table 4. All samples were analyzed in triplicate, along with blanks to check for any loss or cross contamination. For quantitative purposes, the external calibration procedure was carried out with the help of multielemental standard solutions. For building up six point calibration plots, six different concentrations were evaluated per analyte, and each point resulted from triplicate extractions and analyses [102]. For method validation, a linear least-square regression analysis of the calibration graphs was performed to check for the linearity between the instrumental response and the nominal concentration of each elemental standard. The intra-assay and interassay variabilities were determined by quantifying three replicates on the same day and six consecutive days, respectively.


**Table 4.** ICP-MS operating conditions applied to elemental analysis.
