The quality of the quantitative information in single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS) depends directly on the number concentration of the nanoparticles in the sample analyzed, which is proportional to the flux of nanoparticles through the plasma. Particle number concentrations must be selected in accordance with the data acquisition frequency, to control the precision from counting statistics and the bias, which is produced by the occurrence of multiple-particle events recorded as single-particle events. With quadrupole mass spectrometers, the frequency of data acquisition is directly controlled by the dwell time. The effect of dwell times from milli- to microseconds (10 ms, 5 ms, 100 μs, and 50 μs) on the quality of the quantitative data has been studied. Working with dwell times in the millisecond range, precision figures about 5 % were achieved, whereas using microsecond dwell times, the suitable fluxes of nanoparticles are higher and precision was reduced down to 1 %; this was independent of the dwell time selected. Moreover, due to the lower occurrence of multiple-nanoparticle events, linear ranges are wider when dwell times equal to or shorter than 100 μs are used. A calculation tool is provided to determine the optimal concentration for any instrument or experimental conditions selected. On the other hand, the use of dwell times in the microsecond range reduces significantly the contribution of the background and/or the presence of dissolved species, in comparison with the use of millisecond dwell times. Although the use of dwell times equal to or shorter than 100 μs offers improved performance working in single-particle mode, the use of conventional dwell times (3-10 ms) should not be discarded, once their limitations are known.
A methodology based on Asymmetric Flow Field-Flow Fractionation (AsFlFFF) coupled with UV-Vis absorption spectrometry and ICP mass spectrometry (ICPMS) has been developed and applied to the study of silver nanoparticles (AgNPs) and dissolved species of silver in culture media and cells used in cytotoxicity tests. The effect of a nano-silver based product (protein stabilized silver nanoparticles ca. 15 nm average diameter) on human hepatoma (HepG2) cell viability has been studied. UV-Vis absorption spectrometry provided information about the nature (organic vs. nanoparticle) of the eluted species, whereas the silver was monitored by ICPMS. A shift towards larger hydrodynamic diameters was observed in the AgNPs after a 24 hour incubation period in the culture medium, which suggests a "protein corona" effect. Silver(I) associated with proteins present in the culture medium has also been detected, as a consequence of the oxidation process experimented by the AgNPs. However, the Ag(I) released into the culture medium did not justify the toxicity levels observed. AgNPs associated with the cultured HepG2 cells were also identified by AsFlFFF, after applying a solubilisation process based on the use of tetramethylammonium hydroxide (TMAH) and Triton X-100. These results have been confirmed by transmission electronic microscopy (TEM) analysis of the fractions collected from the AsFlFFF. The effect of AgNPs on HepG2 cells has been compared to that caused by silver(I) as AgNO3 under the same conditions. The determination of the total content of silver in the cells confirms that a much larger mass of silver as AgNPs with respect to AgNO3 (16 to 1) is needed to observe a similar toxicity.
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