2.1. Alkaline Digestion and Single-Particle Inductively Coupled Plasma Mass Spectrometry (spICP-MS) Analysis
Different brands of canned tuna (five), mackerel (four), anchovy (three), and clam (three), among the best-selling and low-cost brands, were purchased from the main Italian supermarket chains in the city of Catania, (Italy), in the period between June and July 2019, and stored at −80 °C until analysis. For each brand of seafood product, we also chose to purchase three different batches, which were extracted and processed in triplicate. Thus, we performed a total of 45 extractions for canned tuna, 36 for canned mackerel, 27 for canned anchovy, and 37 for canned clam.
Assessment of TiO
2 was performed using a NexION
® 350D (Perkin Elmer, Waltham, MA, USA) with the Syngistix Nano Application software (Perkin Elmer, Waltham, MA, USA). The instrumental operating conditions for the determination of TiO
2 are listed in
Table 1. The dwell time used was chosen on the basis of other studies [
27,
31].
The new emerging technique of single-particle inductively coupled plasma mass spectrometry (spICP-MS) allows the determination of particle number-based concentration, with rapid simultaneous characterization of the elemental composition, number of particles, size and size distribution, and dissolved concentration. Furthermore, by modifying the integration window, it is possible to collect data related to a specific size distribution. Accordingly, for each sample, we captured data on the total TiO2 particles (Ps-Tot) and TiO2 nanoparticles (NPs < 100 nm) only.
A titanium nanoparticle stock solution was prepared from a TiO2-NP standard (60 nm TiO2 nanopowder, rutile, 99.9%, AEM) purchased from Nanovision (Brugherio, MB, Italy), while a Ti ion standard (1000 mg/L, CPAchem) was used for the spICP-MS calibration of titanium.
To support the quality of spICP-MS measurements, the particle size distribution of the powder TiO2-NPs standard was assessed as follows: a TiO2-NP stock suspension (215 ng/L or 4.5 × 105 particles/mL) was prepared in ultrapure water and dispersed for 30 min at 37 °C using an ultrasonic bath, to maximize a homogeneous dispersion. The transport efficiency (TE%) was calculated with the certified reference material PELCO (Ag-NPs, 39 ± 5 nm, 110 ng/L, monitoring m/z 107) under the same instrumental conditions as the samples, obtaining a value of TE% 2.54. This solution gave a TiO2-NP concentration of 4.6 × 105 ± 0.16 × 105 particles/mL (n = 10), in agreement with the particle concentration prepared. TiO2-NPs showed a size range of 44–85 nm with a mean size of 66.2 ± 3.0 (n = 10) and a modal size of 56.6 ± 4.4 (n = 10). The results obtained were in compliance with the size of the TiO2-NP standard supplied by the manufacturer (60 nm).
Before performing the
spICP-MS analysis, an alkaline digestion of the samples was performed using the method described by Gray et al. (2013) [
32]. Approximately 0.5 g of wet sample tissue was weighed in DigiTUBEs (SCP Science, Baie D’Urfé, Québec, Canada) and mixed with 5 mL of tetramethylammonium hydroxide (TMAH, 20%
v/
v), which is a strong organic base capable of digesting tissues and releasing nanoparticles without altering them. A vortex was used, at first, to prevent the tissues from sticking to the walls of the container used for digestion. The TiO
2 extraction was obtained through sonication for 30 min at 37 °C using an ultrasonic bath to accelerate tissue breakdown and prevent particle aggregation. Subsequently, the samples were left to digest for another 24 h at room temperature. At the end of digestion, samples were diluted with MilliQ water (Millipore, Bedford, MA, USA) to 1% TMAH concentration before analysis and 0.1% Triton X-100 to allow the detection of single particles.
All samples and calibration solutions were sonicated for 30 min before being analyzed. To avoid contamination between samples, the system was rinsed with nitric acid (2%, v/v) prior to the measurement. The TiO2-NP standard was also used for the determination of the transport efficiency within the 3–8% range, in agreement with the TE obtained with an Ag certified reference material.
The effect of the extracting solution on the size distribution and particle concentration was studied in triplicate, simultaneously evaluating the TiO2 standard in ultrapure water (n1 = 3) and TMAH 1% (n2 = 3), at the same concentration (215 ng/L or 4.5 × 105 particles/mL) we used for determining the transport efficiency. We obtained a particle concentration of 4.3 × 105 ± 5.0 × 104 in water and 4.9 × 105 ± 3.6 × 104 in TMAH 1%, with recoveries of 97.6% ± 10.5% and 108.8% ± 7.2%, respectively. In addition, the results revealed that the alkaline digestion in TMAH 1% did not affect the TiO2 particle size distribution (mean size and modal size). The mean size of TiO2 in ultrapure water (63.8 nm ± 3.0) and mean size of TiO2 in TMAH 1% (61.1 nm ± 4.3) were statistically homogeneous according to a two tailed t-test (p = 95%; n1 + n2 − 2) (tcalculated = 1.5 < ttabulated = 2.8). Moreover, the modal size was statistically homogeneous in ultrapure water (57.6 ± 3.2) and in TMAH 1% (56.0 ± 4.0) (tcalculated = 0.88; ttabulated = 2.8).
The limit of detection (LOD) and the limit of quantification (LOQ) were calculated by analyzing 10 alkaline extract blanks, in the same analytical condition of the samples, on the basis of the mean ± 3 SD and the mean ± 10 SD criteria of the number of particles/mL obtained, respectively. The LOD was 1.3 × 103 particles/mL, while the LOQ was 2.5 × 103 particles/mL. Referring to the sample weight and digestion volume used, these values were equivalent to 2.6 × 105/g and 5.0 × 105/g, respectively.
In addition, the LOD in size (LODnm) was estimated at 35 nm using Equation (1) [
26,
33].
where
is three times the standard deviation of the counts/dwell time of alkaline blanks (1% TMAH),
R is the slope of the calibration curve of ionic Ti solutions,
fa is the mass fraction of the analyzed metallic element in the TiO
2 NPs, and
ρ is the density of the TiO
2 NPs.
For each batch of analysis, a quality control was performed with analytical recovery before and after spiking with 60 nm TiO2-NPs at a concentration of 5 µg/L, corresponding to a concentration of 1.1 × 106 parts/mL. The values (range 90–95%) were calculated for the whole size distribution by dividing the TiO2-NP concentration by the TiO2-NP concentration found in the solution of TiO2-NPs used for spiking and multiplying by 100.
Accuracy was tested using a laboratory-fortified matrix (LFM) with a seafood sample spiked with 5 µg/L of TiO2-NPs (60 nm). An LFM was processed at each batch of digestion, obtaining a concentration of 9.8 × 105 ± 4.6 × 105 particles/mL (1.9 × 108 ± 9.3 × 107 particles/g) and a recovery range of 87–121% (n = 5). The measured mean size of TiO2-NPs in alkaline digested samples was 64.2 ± 5.1 nm.
2.2. Acid Digestion and ICP-MS Analysis of Total Ti
For the determination of total titanium, the same samples were processed by acid digestion. Aliquots of 0.5 g of wet samples were weighed in Teflon reactors using an analytical balance (Mettler Toledo) and then digested in a microwave oven (Ethos, TC, Milestone, Sorisole (BG), Italy), by adding 6 mL of 67% super-pure nitric acid (HNO
3; Carlo Erba, Italy) and 2 mL of 30% hydrogen peroxide (H
2O
2; Carlo Erba, Italy) for 1 h at 80 °C. After acid digestion, all samples were diluted to 50 mL with ultrapure water and were filtrated through a 0.45 µm membrane filter before analysis. Total Ti was quantified with the same inductively coupled plasma mass spectrometer (ICP-MS NexION
® 350D, Perkin Elmer, Waltham, MA, USA) in standard mode, using the standard addition technique covering the concentration from 0 to 50 µg/L. Instrumental condition for Total Ti determination are showed in
Table 2.
A single-element standard solution of Ti (1000 mg/L in 5% di HNO3, 0.5% HF) was purchased from CPAchem. Standards for instrument calibration were prepared in the same acid matrix, and yttrium (Y) was used as an internal standard to verify the accuracy.
To verify if acid digestion of the samples allowed the total dissolution of TiO
2 particles and then the quantification of total Ti, we conducted a preliminary acid digestion of 4 µg/L ionic Ti (
n = 6), canned clam (
n = 6), and canned tuna (
n = 6). The quantification of TiO
2 particles was detected using Syngistix Nano Application software, and the resulting background signals, expressed as particles/mL, are shown in
Table 3.
The threshold value of TiO2 particles was estimated as the mean + 3 SD given by ionic Ti, which resulted in 1029 particles/mL. Digested samples of canned clam and canned tuna did not show background values of TiO2 particles higher than the calculated threshold, demonstrating the effectiveness of the acid digestion in dissolving all TiO2 particles.
The LOD and LOQ were calculated by analyzing 10 acid extract blanks according to the mean ± 3 SD and mean ± 10 SD criteria, respectively. They resulted 0.06 and 0.16 mg/kg, respectively.
For each batch of analysis, a quality control was performed with analytical recovery after spiking with 20 µg/L of ionic Ti.
Accuracy was tested using a laboratory-fortified matrix with a seafood sample (use of ionic Ti at 20 µg/L) processed at each batch of digestion, obtaining a recovery range of ionic Ti from 92–115%.