Trace Element Analysis in Biological Samples

Cu content of cell lysates and media samples was measured using a bench-top total reflection X-ray fluorescence (TXRF) spectrometer (S2 Picofox™, Bruker Nano GmbH, Berlin, Germany). As internal standard 1 mg/mL Yttrium (Merck/Millipore) was used. 10 μL of each sample were placed on siliconized quartz glass carriers and dried at 40 °C. Samples were measured in duplicates for up to 500 s. Cu and Se content in liver and colon tissue and Se content of HepG2 cells were determined using ICP-MS/MS. Preparation of samples was described previously [29 (link)]. Briefly, samples were weighted into PTFE microwave vessels. HNO₃ (65% (v/v), Suprapure®, Merck/Millipore), H₂O₂ (30% (v/v), Sigma-Aldrich/Merck), rhodium (Rh) as internal standard, and ⁷⁷Se as isotope dilution standard were added before digestion using a Mars 6 microwave digestion system (CEM, Kamp-Lintfort, Germany). After digestion, samples were diluted to achieve final concentrations of 2.93% (v/v) HNO₃, 10 μg/L⁷⁷Se, and 1 μg/L Rh. The samples were measured using ICP-MS/MS (8800 ICP-QQQ-MS, Agilent Technologies) and analyzed as described earlier [29 (link)]. Certified reference materials, namely fish muscle (ERM BB-422) and pig kidney (ERM BB-186) were used as quality control of digestion and to cross validate TE analysis using TXRF and ICP-MS/MS.