Evaluation of THP-1 zinc content following exposure to zinc oxide nanoparticles by inductively coupled plasma mass spectrometry (ICP-MS)

THP-1 cells were seeded at 5 × 10⁵ cells/ml to 12-well plates. Cells were treated with 0.1 µg/ml zinc oxide 35+, 50+, and 100 + nanoparticles for a period of 24 h. Untreated control cells were prepared in parallel. Cells were collected, washed three times with PBS, and subsequently lysed using 0.5 ml of RIPA buffer and stored at − 80 °C.

Samples were diluted 1:20 with ultrapure water (18 MΩ cm, Veolia Water Technologies, High Wycombe, U.K.) containing germanium (Ge) as an internal standard. Two independent replicates were prepared for each sample. For quality purposes and to assess method accuracy, two solutions containing known amounts of inorganic Zn (~ 2 and 4 µg/kg Zn) were prepared in RIPA buffer to mimic lysis conditions.

Quantification was performed via external calibration using commercial Zn and Ge calibration standards from an accredited supplier (Romil, UK). Zn calibration standards were prepared matching sample conditions in ultrapure water containing Ge as an internal standard, to correct for any instrumental drift or ionisation effects. The external calibration ranged from 0.1 to 10 µg/kg of Zn and ~ 9 µg/kg of Ge. Calibration curves achieved correlation coefficients of at least 0.995.

Samples analysis was performed using a collision/reaction cell ICP-MS 8800 ICP-MS/MS (Agilent Technologies, UK) in helium (He) gas mode. The samples were introduced into the plasma via a MicroMist quartz concentric nebuliser, operating in pumping mode at 0.1 rps, and a Scott double pass spray chamber cooled to 2 °C. The instrument was tuned prior to analysis for optimum signal intensity and stability, with typical operating parameters provided in Additional file 1: Table S1. During analysis, up to six samples were bracketed by 2 blank measurements and a “check” standard, typically the middle calibration standard, to ensure there was no drift.

The LOD/LOQ was evaluated using at least 6 measurements of the reagent blank in the analytical run. The LOD and LOQ were calculated as the mean blank concentration plus 3 and 10 times respectively, the standard deviation of the blank measurements. All sample results were corrected for individual dilution factors. Total Zn mass fractions (µg/kg) are reported for isotope ⁶⁶Zn (27.9 % abundance). Although this isotope is less abundant than ⁶⁴Zn (48.6 %), detection of the latter is likely to be affected by spectral interferences, especially ¹⁶Ar¹⁴N₂+ and ⁶⁴Ni. Results obtained for the other isotope of Zn (⁶⁷Zn, 4.1 %) were in good agreement with those reported. The reported LOD and LOQ for ⁶⁶Zn were based on an average 19-fold dilution of the samples.