Oneneb nebulizer

Purified proteins (0.1 – 0.5 mg/ml) were dialyzed three times against 10 mM Tris·HCl pH 8.0 in deionized (mQ) water in the presence of 0.5% Chelex-100 (Bio-Rad) to remove trace metal ions from the buffer. An axial ICP-AES 720-ES spectrometer (Agilent Technologies, USA) was used for measurements with a low flow axial quartz torch with 2.4 mm inner diameter injector tube (Glass Expansion, Australia), a double-pass glass cyclonic spray chamber (Agilent Technologies), a OneNeb nebulizer (Agilent Technologies, USA), and a Trident Internal Standard Kit (Glass Expansion). Samples were introduced manually to reduce washing volume, without preliminary digestion or dilution. A Sc solution (20 ppm) internal standard was added to increase the accuracy of measurements. Results were collected and processed by ICP Expert software 2.0.5 (Agilent Technologies). An ICP-AM-6 standard solution, 1000 ppm (High Purity Standards) was used for calibration in the range 10–200 ppb.

The hyphenated technique, HPLC-ICP OES was previously described in details [13 (link)] and it was used to determine Fe(II) and Fe(III). The HPLC system was made up of a Shimadzu LC-10AT HPLC pump and a Dionex IonPac CS5A cation-exchange HPLC column (250 mm × 4.0 mm i.d., resin particle size 5 µm, Thermo Fisher Scientific, Waltham, MA, USA). The outlet of the cation-exchange HPLC column was directly connected (avoiding the peristaltic pump) with the nebulizer sample inlets. Peristaltic pump of ICP-OES system was used to drain the waste only. MSIS was applied as a conventional cyclonic spray chamber with OneNeb nebulizer (both Agilent). The detector of the hyphenated technique was Agilent 5110 ICP-OES (Agilent). The operating conditions were placed in Table 2. The sample was analyzed 3 times (n = 3). All DLs and QLs were calculated as described in the Section 2.4 and the results were presented in Table 3. The standard addition method was used for accuracy studies. Recoveries were in the acceptable range (80–120%) and full results were presented in Supplementary Materials (Table S1). The uncertainty for the complete analytical process (including a sample preparation) was at the level of 20%.

The collected samples were acid digested and elemental distribution of the materials in different in vitro test compartments measured by inductively coupled plasma optical emission spectrometry using an ICP-OES 5100—vertical dual view apparatus coupled with OneNeb nebulizer (Agilent Technologies, Santa Clara, CA, United States). Digestive procedure was performed adding 0.2 ml of hydrogen peroxide (H₂O₂ 30 wt % in water), 0.2 ml of sulfuric acid (H₂SO₄ 96%), 0.2 ml of phosphoric acid (H₃PO₄ 85%) and 0.2 ml of nitric acid (HNO₃ 65%) in to 0.5 ml sample and adding 1.5 ml of MilliQ water. The treated samples were ultrasonicated for 10 min in an ultrasonic bath. Calibration curves were obtained with 0.01, 0.05, 0.1, 1.0, 10.0, and 50.0 µg/ml standards prepared in RPMI, using the same digestive procedure applied to samples. Standards were treated adding a 10% v/v of sulfuric acid, phosphoric acid and of nitric acid, and ultrasonicated for 10 min, as well.