Multi element environmental calibration standard

Whole blood or serum samples were collected from all participants and measured by an inductively coupled plasma mass spectrometer, Agilent ICP-MS 7900 (Agilent Technologies, Tokyo, Japan), equipped with standard Ni sampling and skimmer cones and an Ultra High Matrix Introduction system. Calibration standards were prepared from an Agilent multi-element environmental calibration standard (p/n 5183-4688) containing 10 ppm each of Cu, Mn, Mo, Se, and Zn. The internal standard (Sc, Ge, Rh, In, and Bi) for the quantitative analysis was made in 1% HNO₃ and 0.5% HCl for the diluted sample. Triton C-X-100, Butanol, EDTA, ammonium hydroxide, and water were added to the alkaline solution. Dilution and sample preparation were performed under a clean hood to prevent contamination by atmospheric particulates. An amount of 100 μL of the standard was added to a 15 mL polypropylene tube, and then 22 mL of diluent was added with a dispenser. Next, 100 μL of samples and the control were dispersed in the dispensed diluent. Diluted samples were vortex-mixed and analyzed with Agilent ICP-MS.

Three technical replicates of reconstituted TdAA15A (after copper loading and gel filtration) were transferred to a digestion vessel. The samples were digested using 1 mL of nitric acid (70%, trace metal grade). The quartz vials were transferred to a microwave digestion system (Ethos Up) and heated to 200 °C (sealed vessel) for 15 min, then allowed to cool to room temperature. Once cooled, the reaction mixture was transferred to a 10-mL volumetric flask and diluted to volume using deionised water (18 MΩ). Copper concentration was measured using an Agilent 7700x inductively coupled plasma-mass spectrometer (ICP-MS). Seven calibration standards were prepared using certified reference standards (multi-element environmental calibration standard, Agilent, Part number 5183–4688). Calibration curves had r₂ values of 0.998 or better. De-ionized water (18 MΩ) was used as a blank. The values obtained for the three protein samples were corrected against three technical replicates of the negative control (buffer only).