Standards and Reagents
A standard 999 ± 2 µg L− 1 dissolved Zn stock solution in 5% HNO3 (Sigma-Aldrich, Saint Louis, MO, USA) was used to prepare an intermediate 100 µg L− 1 solution. The calibration curve was prepared in 15 mL Falcon flasks using successive dilutions in ultrapure water.
Monodisperse ZnONP suspensions were prepared from commercially available solutions. ZnONPs measuring < 100 nm particle size (DLS) > 35 nm avg.part size (APS), 50 wt. % in H2O. (Sigma Aldrich, Missouri, EUA). After dilution and before each analysis, the suspensions were sonicated and vortexed for 1 min.
AuNP standard solution of nominal size of 50 nm, supplied by NanoComposix (San Diego, CA, USA), was used to validated the TE, it is not required to use de nanoparticle the same element that will be analysis (21).
For the quality assurance of the result, Water 1643F acquired from the National Institute of Standards and technology (Nist) was used for evaluating the accuracy, precision, and recovery study.
Instrumentation
The experiments were performed on a NexION 300D ICP-MS (Perkin Elmer, USA), equipped with a concentric nebulizer type Meinhard, glass cyclonic nebulizer chamber, cone, skimmer, and nickel hyper-skimmer. ICP-MS instrumental and data acquisition parameters are listed in Table 1. Although 67Zn has a lower natural abundance than the other Zn isotopes, it was chosen to avoid 50Ti16O interference that occurs with the 66Zn isotope and is found in greater abundance (22, 23).
In the ICP-MS technique, only a fraction of the nebulized suspension effectively reaches the plasma (1–15%) when using conventional systems, and this, too, happens in the single particle mode (24). The precise determination of this fraction, defined as transport efficiency (TE), is critical for correctly determining both particle number concentration and size (25). The TE was calculated by the particle frequency (TEF) methods using an AuNP standard solution of nominal size of 50 nm, which was sonicated for 1 min and diluted 100-fold in deionized water (Milli-Q Advantage, Molsheim, France) to a final nominal concentration of 1 × 105 particles mL-1, supplied by NanoComposix (San Diego, CA, USA). The interday repeatability of TE was calculated, and the mean was used for validation. The variations found can be strongly influenced by the daily optimization procedures of the ICP-MS instrument.
In developing the methodology, we applied two complementary approaches to ensure the precision and efficacy of the analytical process. The waste collection technique was employed to ensure accuracy in the volume of sample aspirated, a critical factor for the reliability of nanoparticle quantification results. Concurrently, the particle frequency approach was utilized to optimize the transport efficiency (TE), ensuring our SP-ICP-MS system could detect and quantify nanoparticles with the highest precision possible. Although these strategies serve different purposes, both are fundamental to the integrity of our results, demonstrating the rigor and robustness of our analytical methodology (24, 26).
The Zn total (t-Zn) concentration was determined by inductively coupled plasma optical emission spectrometry – ICP OES – model Optima 8300 (Perkin Elmer, USA) equipped with a GemConesTM nebulizer, cyclonic glass nebulizer chamber.
Table 1
Default single particle inductively coupled plasma mass spectrometry instrumental and data acquisition parameters.
Instrumental parameters | | |
RF Power | 1400 W | |
Argon gas flow rate | | |
Plasma | 18 L min− 1 | |
Auxiliary | 1.2 L min− 1 | |
Nebulizer | 1 L min− 1 | |
Data acquisition parameters | | |
Measurement unit | Standard | Single particle detection |
Point per spectral peak | 1 | 1 |
Sweeps | 20 | 1 |
Dwell time | 50 ms | 50 µs |
Readings per replicate | 1 | 2000.000 |
Integration time | 1 s | 100 s |
Method Parameter | Zn | |
Isotope (amu) | 67 | |
Density (g/cm3) | 5.61 | |
Mass Fraction (%) | 80.31 | |
Ionization Efficiency (%) | 100 | |
Sample preparation
The lack of reference certified materials for NP in different matrixes, led us to use recovery tests to fulfill the validation requirements. A spiking control sample with NP standards and ion dissolved standards were used to evaluate the accuracy and precision. A sunscreen sample that declared on the label no ZnONP was used as negative control. 0.30 g of the sample was weighed (triplicate) and submitted to two hours ultrasonic treatment, followed by centrifugation at 5,000 rpm for 30 min, and filtration through 0.22 µm pore membranes. After filtration, each sample was sonicated and vortexed for 1 min. The same extraction process was applied to a blank solution containing only ultrapure water.
The t-Zn determination was made, weighing approximately 0.5 g of ZnONP suspensions in triplicate. The samples were transferred to Teflon tubes containing 2 mL of deionized water (Millipore, Brazil), 2 mL of 65% (w/v) nitric acid (Merck, Germany), and 2 mL of 30% hydrogen peroxide (v/v) (Merck). The sample digestion was made using microwaves in an enclosed high-pressure system, SpeedWave microwave (Berghof, Germany), and after chilling, the samples were transferred to 50 mL volumetric flasks (27).
Preparation of the analytical curve
Five analytical curves were prepared using a Zn standard dissolved in a concentration range from 1 to 20 µg L− 1. The samples were determined in SP-ICP-MS mode, and the analytical curve of dissolved Zn was crafted considering the relationship between the signal strength (counts) and mass per event (µg/event), converted by equipment software.
Selectivity
The selectivity of the technique was determined by matrix effect (standard addition). Two distinct groups containing Zn ions were prepared, a group of the matrix of interest (group 1) with known concentration of the analyte, and another group made up of water and the analyte (group 2) (25).
Limited of detection
ZnONP identification and quantification by SP-ICP-MS depends on two factors: (i) the size of the NP, which must be large enough to generate several ions detectable by the spectrometer, and (ii) the numerical NP concentration, which must be high enough to allow counting a minimum number of events (24). Therefore, three LODs are calculated: the LOD size (LODd) for the diameter of NPs, the concentration of the number of NPs LOD (LODp), and LOD for ion dissolved zinc (Zn(i)). The limits of detection were obtained by reading of ten solutions independent of the blank and calculated according to studies published by Laborda et al.,2020, using parameters such as TE, dwell time, flow rate, time integration, sensibility of ion concentration (slope of concentration curve), count number of particle and mean of baseline signal (21, 25, 26, 28).
The limit of quantification (LOQ) for zin(i) was obtained experimentally, defined as the first point of the calibration curve (20).
Accuracy and precision
The method's accuracy was determined by the analysis of ZnONP standards by two different techniques: ICP OES and ICP-MS. t-Zn results were evaluated by application of Student's t-test. The results indicated there is no significant difference between the results (p > 0.05). The average ZnO concentration is equal to 44.8 g L− 1. The theoretical number of nanoparticles/mL (3.7 x 1013 particles mL− 1) was determined using the average ZnO concentration. It was possible to calculate the declared average size of the certified standard (75nm) (23).
To assess the method's accuracy and precision, a sample of Nist 1643F and sunscreen were prepared to contain ZnONPs and dissolved Zn. The Zn(i) concentration was chosen to have a concentration in the middle of the calibration curve, and the ZnONP concentration was close to the LOD. The solutions were analyzed five times by SP-ICP-MS, and the average amount of particles, size, and Zn (i) concentration for each sample was compared with the theoretical results.
Statistical analyses
Descriptive statistics were obtained using Microsoft Excel, including arithmetic mean, median, standard deviation (SD), Student's t-test, and analysis of variance (ANOVA). The uncertainty was estimated by adopting the 'bottom-up' method, which starts with identifying and characterizing the individual sources of uncertainty and then combines them to obtain the total uncertainty. The main sources are represented in Fig. 1, where the repeatability of the methodology is associated with random effects and was verified by the standard deviation of the response and quantified from repeated experiments and verified by the standard deviation of the response; sample preparation is associated with calibration uncertainties obtained from the calibration certificates of the volumetric flask and analytical balance used; the uncertainty of the standards is associated with the uncertainty of the stock solution purchased and the calibration certificates of the volumetric flask and pipettes used, and the uncertainty of the calibration curve is associated with the uncertainty of the angular coefficient (30). Once the coverage factor k (k = 2) was set at a 95% confidence level and the final combined uncertainties were estimated, the final expanded uncertainty was calculated (18, 19).