8800 triple quadrupole icp ms

Caco-2 cells (1 × 10⁵), HCT116 cells (5 ×10⁵) or HCEC (2 × 10⁵) were grown in 6 cm dishes and treated with hemin or ferric chloride for 0.25, 1, 8 and 24 h. Cells were trypsinized and harvested by centrifugation. The pellets were then digested in 0.5 ml H₂O₂/HNO₃ (1:1) overnight at 95 °C. The residue was dissolved in 1.5 ml 10% HNO₃ with 0.5 µg/l Rh (Merck, Darmstadt, Germany) as internal standard and diluted if required. Quantification was performed with an external calibration curve ranging from 0.5–100 µg/l Fe (Spetec, Erding, Germany). The measurements were conducted with a tandem mass spectrometer (8800 triple quadrupole ICP-MS, Agilent Technologies, Böblingen, Germany) equipped with two quadrupols (Q1 and Q2) as well as a collision and reaction cell with O₂ (0.3 ml/min) as collision gas. Quantification was carried out directly using the ⁵⁶Fe⁺ ion (Q1 and Q2 mass-to-charge (m/z) ratio 56) or indirectly using the ⁵⁶Fe¹⁶O⁺ ion (Q1 m/z 56 and Q2 m/z 72).

Iron and molybdenum contents of MoFe- and Fe-protein samples were quantified using an Agilent 8800 Triple Quadrupole ICP-MS in both oxygen mode (mass shift) and helium mode (kinetic energy discrimination) running Agilent MassHunter software. Operating parameters were determined following standard protocols recommended by the manufacturer that determined flow rates of 0.4 and 4.2 mL/min for oxygen and helium, respectively. In oxygen mode, iron and molybdenum were determined using 56 to 72 m/z and 95 to 127 m/z, respectively. In helium mode both iron and molybdenum were determined in MSMS mode, 56 to 56 and 95 to 95, respectively.
Standard mixtures containing iron and molybdenum (Periodic table mix 1 for ICP, product #92091; molybdenum standard for ICP-MS, product #04488) were prepared in 5% nitric acid spanning 0–100 μg/L for Periodic table mix 1 and 0–50 μg/L for the molybdenum standard. Ten to 30 μL of protein sample ranging from 3 to 18 mg/mL was diluted into 285 μL of 70% nitric acid and incubated at 50 °C for 3 h to release metals bound to the protein. Samples were then diluted into a final volume of 10 mL MilliQ water. The iron and molybdenum concentrations were determined using ⁵⁶Fe and ⁹⁵Mo isotopes and normalized to the MoFe-protein concentration in the sample.

Potentially hazardous toxic metals (As, Cd, Pb, Hg) were determined using an Agilent 8800 Triple Quadrupole ICP-MS supplied with an ASX500 autosampler (Agilent Technologies, Tokyo, Japan) in helium acquisition mode. The sample introduction system consisted of an integrated peristaltic pump set to 0.1 rps, which aspirated the sample into a concentric nebulizer. The operating conditions of ICP-MS were 1550 W of RF power, 1 L/min of carrier gas flow and 0.7 mL/min of He flow. The instrument was calibrated in the range of 0.5 to 25 µg/L for As, Cd and Pb, and from 0.1 to 10 µg/L for Hg. The calibration standards were prepared starting from 10 µg/mL standard solutions, purchased from Agilent Technologies. Prior to the sample measurements, an amount of 1 g of nutraceutical was weighed in a PFA digestion vessel, over which 8 mL of concentrated HNO₃ (Suprapur^®, Merck, Darmstadt, Germany) was added. The PFA tube containing the sample was subjected to a microwave-assisted digestion treatment (Ethos UP, Milestone Inc., Sorisole, Italy). The obtained solution was further quantitatively transferred to a 25 mL volumetric flask and subsequently diluted up to a mark with Milli-Q water (18 MΩ·cm).

The boiled soup was filtered using a 100-kDa ultrafiltration spin column and precipitated using TCA; the pellet was treated with nitric acid and then analysed using ICP-MS. The concentration of S, Na, Mn, Ti, As, Fe, Ni, Cu, Li, Mg, Al, Si, P, K, Ca, Co, Zn, Rb, and Cs was determined using ICP-MS (8800 Triple Quadrupole ICP-MS; Agilent, CA, USA), following the manufacturer’s instructions.

3-BPA or 4-BPA (40 mg) were suspended in 200 μL water and stirred at 25 °C for 24 h. After stirring, the solution was centrifuged at 5000 rpm for 10 min, and the amount of boron in the supernatant solution was measured by 8800 triple quadrupole ICP-MS (Agilent, Santa Clara, CA, USA). The experiment was repeated twice with n = 3.