Icap 6000

Samples were processed using the MARS 6 Microwave digestion system. Samples (0.5 g of starting material or 5 mL of digest) and 5 mL of concentrated nitric acid were added into reaction vessels and placed into the microwave digester. Digestion of the samples was carried out for an hour. The contents were then transferred into Falcon tubes containing 140 μL of 100 ppm yttrium internal standard, and the volume was made to 14 mL with deionised water. Iron in the samples was read using the inductively coupled plasma optical emission spectrometry (ICP-OES) (Thermo ICAP 6000).
Fractionation of the soluble iron into total bioaccessible Fe (TBF) and low-molecular-weight Fe (LWT) in the digested extracts was carried out by centrifugation and ultrafiltration, as described by Powell et al. [18 (link)]. Aqueous suspensions (0.5 mL) were centrifuged (1000 rpm, 5 min), and the supernatant represented the total bioaccessible Fe fraction during in vitro digestion. To separate the low-molecular-weight Fe fraction, a proportion of the supernatant was ultrafiltered through AMICON ULTRA 3 kDa molecular weight cut-off columns (Merck-UFC500396) (1000 rpm, 5 min). Iron concentrations of samples were determined in ICP-OES (Thermo ICAP 6000). The TBF and the LMW were calculated (after subtracting 0.84 ± 0.02 µg/g Fe present in the digestive enzymes) as follows:

Fifty mg of dried and milled wheat grain flour was taken to be digested by (2 mL) nitric acid (HNO₃ 69%, Bernd Kraft GmbH, Germany). The digestion process was completed using a high-performance microwave reactor (UltraClave IV, MLS, Leutkirch im Allgäu, Baden-Württemberg, Germany). All digested samples were filled up to 15 mL final volume with de-ionized distilled (Milli-Q) water (Milli-Q^® Reference System, Merck, Germany). Element standards were prepared from Bernd Kraft multi-element standard solution (Germany). Fe as an external standard and Yttrium (Y) (ICP Standard Certipur^® Merck, Germany) were used as internal standards for matrix correction. Fe concentrations were measured by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES, iCAP 6000, Thermo Fisher Scientific, Dreieich, Germany) combined with a CETAC ASXPRESS™ PLUS rapid sample introduction system and a CETAC autosampler (CETAC Technologies, Omaha, NE, USA).