Trace metal grade hno3

Zebrafish samples from vehicle, 0.03, 0.3, and 3 ppm Ag-NP exposure groups were analyzed at 3 and 5 dpf to quantify tissue concentrations of Ag. Samples were analyzed in triplicate with 22 to 26 larval zebrafish/sample obtained from 3 separate spawnings. Zebrafish samples, triplicate method blanks (200 µL 18.2 MΩ/cm water), and triplicate NIST1640a digestion quality control standards (200 µL NIST1640a Trace Elements in Natural Water - National Institute of Standards and Technology) were digested by adding 30 µL concentrated TraceMetal Grade HNO₃ (Fisher Scientific) and 100 µL concentrated TraceMetal Grade HCl (Fisher Scientific) then heating in a hot block that was ramped up to 95 ºC over 30 min, followed by digestion at 95 ºC for 1 h. 550 µL 30% ULTREX II H2O2 (J.T. Baker) was added incrementally to room temperature samples with heating between additions. The following increments were used: 50 µL at 4 ºC for 15 min, 100 µL at 67 ºC for 10 min, 100 µL at 85 ºC for 45 min, 150 µL at 95 ºC for 25 min, and 150 µL at 95 ºC for 1 h. The samples were allowed to cool, then brought to a final volume of 1 mL with 18.2 MΩ-cm water prior to analysis by the Interdisciplinary Center for Plasma Mass Spectrometry at the University of California at Davis (ICPMS.UCDavis.edu) using an Agilent 8900 ICP-MS (Agilent Technologies).

Metal composition was determined using an Agilent 5110 Vertical Dual View Inductively Coupled Plasma - Optical Emission Spectrometer (VDV ICP-OES) (Agilent, Inc.). Cells were resuspended in 5% HNO₃ for manual lysis or in 17.5% HNO₃ for lysis in a Multiwave Go microwave digester (Anton Paar) as described previously.^{79 (link)} Intracellular metal concentrations were determined by comparison with a standard curve created with a 10 μg ml⁻¹ multi-element standard (CMS-5; Inorganic Ventures) diluted with 5% HNO₃ to generate dilutions ranging from 5.00 to 0.01 μg ml⁻¹. To avoid contamination with metals, trace-metal grade HNO₃ was used (Fisher Chemical). Nitric acid was used in the lysis of cells in order to improve the lysis process and to provide greater stability of the samples for analysis by ICP-OES, ensuring that the analytes remained in solution for longer periods of time, which is particularly important with the transition metals as they are prone to hydrolysis. Specific wavelengths analyzed were Mn: 257.61 nm, Fe: 259.94 nm, Zn: 202.548 nm, Cu: 324.754 nm. Results are based on the means and standard deviations from at least three replicates of independent cultures for each strain/growth condition.

All NMR spectra were obtained on a Bruker DRX 500 or Bruker Avance 400 broad band FT NMR spectrometers. ¹¹B chemical shifts were referenced to BF₃•Et₂O (15% in CDCl₃, δ 0.0 ppm) unless otherwise stated. All ICP-OES data were obtained on an Agilent ICP-OES 5100 spectrometer. Samples were diluted in 4% HNO₃ using trace metal grade HNO₃ (Fisher) and LC-MS Grade water (Fisher). Standard solutions were prepared from commercially available 1000 ppm stock solutions of boron (Acros) and iron (Ricca Chemical). Standard addition using a 2 ppm yttrium stock solution was used for all samples. Plots and statistics (k, half-life, etc) for dynamic dialysis and biodistribution experiments were calculated using GraphPad Prism software.