Mars 6

Breast milk samples, a standard reference material (SRM 1953; National Institute of Standards and Technology (NIST)), and sample for method detection limit (MDL) measurement were brought to room temperature by letting them sit at room temperature and gently swirling to avoid frothing, before 100 µL was aliquoted into a 15 mL MetalFree^® centrifuge tube (Labcon, Petaluma, CA, USA) and accurately weighed. To this sample, 3.9 mL of the alkaline dilution solution was added before samples were vortex-mixed and microwaved using the microwave digestion system MARS 6 (CEM Corporation, Matthews, NC, USA). Microwave conditions are described in Table S1. The power of the microwave was determined so as not to exceed the durability of the tube. After bringing the treated sample to room temperature, 4 mL of the detergent solution containing the internal standard was added and mixed by inverting the sample before the sample was visually examined to ensure that there was no particulate matter (Figure S1).

Four heavy metals (lead, cadmium, mercury, and arsenic) were analyzed according to a method of the Food Code [26 ]. Each sample (10 g) was heated in a microwave oven (MARS 6, CEM Corporation, Matthews, NC, USA) at 450 °C until completely carbonized, and then homogenized using a homogenizer (SHG-15D, SciLab Co., Ltd., Seoul, Korea). Subsequently, they were dissolved in 5 N nitric acid solutions to obtain at least 20 mL of experimental solution for each sample. The concentrations (mg/kg) of lead, cadmium, and arsenic were measured by inductively coupled plasma (ICP; Optima 5300 DV; Perkin Elmer, Waltham, MA, USA) in triplicates, whereas 1 g of the sample was analyzed using a mercury analyzer (MA-2; Nippon Instruments Corporation, Tokyo, Japan) to determine the mercury concentration.

Validated atomic absorption spectrometry (iCE 3000 Series, AAS, Thermo Scientific, Cambridge, UK) was used to determine serum calcium concentration.
Sample dissolution was carried out using a microwave accelerated reaction system (MARS 6, CEM Corporation, Matthews, NC, USA). For this purpose, 1 mL of blood serum was collected in a microwave vessel and 7 mL of 69% ultra-pure nitric acid (ROMIL, Cambridge, UK) was added. The vessels were gently mixed and left open for 15 min to facilitate initial sample digestion. The microwave heating program consisted of two stages: increase from ambient temperature to 180 °C in 20 min and then this temperature was maintained for a further 20 min. After cooling, the resulting solutions were transferred to 50 mL volumetric flasks and diluted to 20 mL with ultra-pure water. All analyses were repeated three times. The correct serum calcium concentration range was between 2.25 and 2.75 mmol/L [84 ].

Pi concentration in wheat seeds (7, 14, 21 and 28 DAA), 14 DAA seed tissues (aleurone, endosperm and embryo) and starvation samples (roots and shoots) was measured by molybdate-blue colorimetric method56 . The 0.5 g of tissues in three biological replications was extracted in 0.5 ml of extraction buffer (10 mM Tris, 1 mM EDTA, 100 mM NaCl, 1 mM β-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride, pH 8.0) as described by earlier57 (link). Further, 0.7 ml of assay solution (Ascorbic acid, 10% and 0.42% ammonium molybdate in 1 N H₂SO₄) was added to 0.3 ml of sample solution (or to 0.3 ml of water for the blank) and incubated at 42 °C for 40 minutes. Pi concentrations were detected at the absorbance of 820 nm. For the standard preparations, 1 mM of KH₂PO₄ solution was used at different volumes (0, 5, 15, 20, 25, 30, 35, 40, 45 μL).
For total P analysis, approximately 100 mg powder was digested with nitric acid in the Microwave Reaction System (Mars 6, CEM Corporation, USA). P concentration was estimated in the digested samples using inductively coupled plasma mass spectrometry (ICP-MS; 77006AgilentTechnologies, Santa Clara, CA), following standard protocol.

For mineralogical analyses, fragments of various growth regions (i.e., new-growth from the dorsal margin and older-growth from near the ventral margin) of T. squamosa shell and scute were crushed into a fine powder using a clean, porcelain, pestle and mortar, which was thoroughly rinsed with MilliQ ultrapure water and blotted dry with filter paper in between individual shell preparations to avoid cross-contamination. Approximately 0.5–1.5 mg of each powdered shell sample was then completely dissolved in 10 mL 2% HNO₃ (75% TraceMetalTM Grade, Fisher Chemical, Loughborough, UK) in a microwave reaction system (MARS 6, CEM Corporation, Matthews, NC, USA). Ionic concentrations (i.e., ⁴⁰Ca, ²⁴Mg, ²⁸Si, ³¹P, ³⁹K, ⁵⁵Mn, ⁷⁵As, ⁸⁸Sr, and ¹³⁷Ba) were measured from aliquots of this solution using inductively-coupled-plasma mass spectrometry (ICP-MS, X-Series II, Thermo Scientific, Hemel Hempstead, UK) following the method of Wolf and Adams (2015)³⁴ . When necessary (e.g., for ⁴⁰Ca), 200-fold dilutions of prepared samples were made in 2% HNO₃. Samples were run in triplicate and all elements were analyzed simultaneously. Raw elemental concentration data for ⁴⁰Ca and ⁸⁸Sr were drift-corrected using the sample-standard correction method described by Schrag^{35 (link)}.

Al, B, Ca, Fe, K, Mg, Mn, Na, Sr, and Zn content was measured in dry samples by inductively coupled plasma optical emission spectrometry (ICP-OES) after mineralization of the dry samples with an acid microwave-assisted digestion system (MARS 6, CEM Corporation, Matthews, North Carolina) performed as reported by D’Imperio et al. [34 (link)]. To confirm the accuracy of the measurements, certified reference vegetable material (CRM, NIST tomato leaf 1535a) was analyzed using the same procedure as the rocket and purslane samples.

The concentration of Cu and Zn in 103 formula samples for infants was determined using an atomic absorption spectrometer. The determination was based on the method described by Yaman et al. [16 ]. Sample digestion was carried out using a microwave accelerated reaction system (MARS 6, CEM Corporation, Matthews, NC, USA). Each sample (1 g) was weighed directly into a microwave vessel and filled with 10 mL 69% ultra-pure nitric acid (ROMIL, Cambridge, UK). Vessels were gently mixed and left open for 15 min to allow sample pre-digestion. The microwave heating program consisted of 2 steps; ramping from ambient temperature to 180 °C over 20 min, and then holding the temperature for 20 additional minutes. After cooling, the obtained solutions were transferred to 50 mL volumetric flasks and diluted to 20mL with ultrapure water. The atomic absorption spectrometry method (iCE 3000 Series, AAS, Thermo Scientific, Cambridge, UK) was used to determine the concentration of both Cu and Zn in samples. All analyses were performed in triplicate. The methods were validated by a simultaneous analysis of the reference material, with accuracy for Cu of 94.5% and for Zn of 95.4%.