WO2013164610A1

WO2013164610A1 - Method of determination of titanium levels in tissues

Info

Publication number: WO2013164610A1
Application number: PCT/GB2013/051120
Authority: WO
Inventors: Richard HANDY
Original assignee: University Of Plymouth
Priority date: 2012-05-03
Filing date: 2013-05-01
Publication date: 2013-11-07
Also published as: GB2501738A; GB201207745D0

Abstract

Methods of tissue sample preparation and analysis are disclosed, in which a sample is prepared by digesting the tissue in acid, subsequently adding a non-ionic surfactant and diluting the digested tissue with water, wherein the concentration of the non-ionic surfactant in selected to disperse titanium dioxide nanoparticles present in the diluted sample; and agitating the sample immediately prior to analysis to further disperse the particles. Methods are disclosed wherein the concentration of the non-ionic surfactant in the diluted sample are above the critical micelle concentration. Methods are disclosed wherein the sample is subsequently analysed by inductively coupled plasma optical emission spectrometry (ICP- OES) to determine the concentration of metals including titanium in the form of titanium oxide nanoparticles and/or by inductively coupled plasma mass spertrometry (ICP-MS) to determine the characteristics of titanium dioxide nanoparticles in the sample. In some methods, the same prepared sample may be used for both ICP-OES and ICP-MS.

Description

Method for Determination of itanium Levels In Tissues

The present invention relates to a method for determining the levels of titanium in tissues, and in particular to a method which can be used to analyse nanoparticles and bulk powders comprising titanium.

With the growing development, use and inevitable environmental release of engineered nanomateriais a need exists for methods which enable the routine analysis of concentrations of metal from metallic engineered nanoparticles (NPs) in biological tissues, in toxicology studies such measurements are required in order to confirm and characterise exposure. For example, tissue concentration levels are required fo calculating the b loeon centra ti o factor and the rate of uptake and elimination, as well as aiding in hazard assessment, in food safety, methods for monitoring metals in fish and .shellfish are important.

Such methods need to be safe, reliable, reproducible and able to be carried out with high throughput (for example handling hundreds of samples per day). However the unusual properties of nanoparticles that proves beneficial in their use present technical barriers that make such methods difficult to achieve. Methods have not yet been developed which are suitable for engineered nanomateriais.

I addition to simply determining the total concentration of a specific anaiyte within a tissue sample, it is often also necessary to analyse for trace elements other than the specific anaiyte. In order to fully understand the toxicology of nanoparticles, an analytical method which provides information on both the concentration of elements and the nature of the nanoparticles within a tissue sample is desirable.

Titanium dioxide (Ti0₂) nanoparticles have a number of applications, for instance being widely used in sun block and in pigments. Traditional methods for determining trace meta!s in animal tissues comprise digesting the tissue in acid, followed by analysis by inductively coupled plasma optica! emission spectrometry, or ICP-OES (aiso sometimes known as inductively coupled plasma atomic emission spectrometry or ICR- AES). An example of such a prior art method is described in Shaw, B. J,, Handy, R. D., 2006. Dietary copper exposure and recovery in Nile tilapia oreochromis niloticus, Aquatic Toxicology 76, 111-121. These techniques cannot readily be applied in the analysis of Τί<¾ nanoparticies since they tend to result in very poor ratios of detected titanium to the amount present in Ti(¾ NPs (hereinafter referred to as the recovery ratio). An example of previous analysis of Ti0₂ by ICP-AES is disclosed in Scown, T.M., van Aerie, ft., Johnston, B.D., Cumberland, S,, Lead, J.R., Owen, R._f Tyler, C.R,, 2009. High Doses of Intravenously Administered Titanium Dioxide Nanoparticies Accumulate in the Kidneys of Rainbow Trout but with no Observable Impairment of Renal Function. Toxicological Sciences 109, 372-380. The recovery ratio reported therein was less than 30%.

One method of improving the recovery ratio would be to fully dissolve the Ti0₂ by the use of strong acids, such as hot concentrated sulphuric acid or hydrofluoric acid. Such methods require specialised equipment and, particularly in the case of hydrofluoric acid, are extremely dangerous and are therefore not appropriate for timely and cost effective routine analysis of a large number of samples.

Scown et ai supra discloses the use of Triton X-100 to disperse nanoparticies but the recoveries are unacceptably low. Scown reports a 0.2% by volume Triton X-100 concentration in the analysed sample (200μί of 10% Triton X-100 in a 10ml sample).

The use of inductively coupled plasma mass spectrometry (ICP-MS) for single particle characterisation of water samples has recently been reported, for instance in Laborda, P., Jimenez-Lamana. J., So/ea, Castillo, J.R., 2011, Selective identification, characterization and determination of dissolved $ilver{i) and silver nanoparticies based on single particle detection by inductively coupled plasma mass spectrometry. Journal of Analytical Atomic Spectrometry 26, 1362-1371. This document describes the use of ICP-MS in which a sample containing particles is introduced to the instrument, and single particles are detected, following ionisation, as a single pulse at the detector. In principle the particle size, and size distribution can be evaluated by correlation to the pulse intensity. The mass spectrometer detector allows the composition of the particles to be determined. Single particle ICP-MS has previously been carried out only on relatively pure water samples,.

A method that enables characterisation of single particles from tissue samples is desirable. Furthermore, a method that enables the accurate routine analysis of a tissue sample to determine mass concentrations of titanium from titanium oxide nanoparticfes is desirable, it is further desirable that a method is provided, in which a single prepared tissue sample can be analysed both to determine mass concentrations of titanium from titanium oxide nanoparticies and to determine the characteristics of the nanoparticfes, such as the relative particle size distribution.

According to a first aspect of the invention there is provided a method of preparing a tissue sample for analysis of titanium levels therein, comprising: digesting the tissue sample in acid; subsequently adding a non-ionic surfactant and diluting the digested tissue with water, wherein the concentration of the non-ionic surfactant is selected to adequately disperse titanium oxide nanoparticies present in the diluted sample; and agitating the sample immediately prior to analysis to further disperse the nanoparticies.

According to a second aspect of the invention, there is provided a method of analysing tissue, comprising preparing a sample according to a first aspect of the invention; and promptly following agitation, analysing the sample by using inductively coupled plasma mass spectrometry to determine the characteristics of titanium oxide nanoparticies in the sample, According to a third aspect of the invention, there is provided a method of analysing tissue, comprising; preparing a sample according to a first aspect of the invention; and promptly following agitation, analysing the sample by using inductively coupled plasma optical emission spectrometry to determine the concentration of metals including titanium in the form of titanium oxide nanoparticies,

According to a fourth aspect of the invention, there is provided a method of analysis tissue, comprising: preparing a sample according to a first aspect of the invention; promptly following agitation, analysing the sample by: using inductively coupled plasma optical emission spectrometry to determine the concentration of metals including titanium in the form of titanium oxide nanoparticies; and using inductively coupled plasma mass spectrometry to determine the characteristics of titanium dioxide nanoparticies in the sample.

The non-ionic surfactant may be present in the diluted sample at concentrations in excess of the critical micelle concentration. The non-ionic surfactant is preferably Triton X- 00. it is preferable for the diluted sample to have a Triton X-100 concentration of between 1 ,5% and 3% by volume. The diluted sample may be placed in a container seiecied to maximise the dispersio of nanoparticies during agitation. The container may preferably be a centrifuge tube, for example a 15ml centrifuge tube.

Agitating the sample may comprise swirling the sample on an orbital shaker. The orbital shaker may rotate at between 130 and 160 revolutions per minute. The sample fS preferably agitated for at least 30 minutes, it is preferable for the analysis to be carried out within less than 5 minutes of agitation. More preferably, the analysis should be carried out within 1 minute of agitation. Agitating may comprise vortexing the sample. The sample may preferably be vortexed immediately prior to analysis.

The acid may be nitric acid. The acid is preferably concentrated nitric acid. The volumetric ratio of the acid and the diluted sample is between 3 and 6.

The analysis b inductively coupled plasma optical emission spectroscopy is preferably calibrated with a matrix matched calibration standard. The calibration standard may comprise titanium metal.

The tissue may be fish tissue. The invention will now be described, by way of example, with reference to the following drawings, in which:

Figure 1 shows the effectiveness of an embodiment of the invention in the recovery of Ti in rainbow trout tissue (gill, intestine, liver) spiked with TOO pg/i T1O2 NPs;

Figure 2 shows recovery of Ti in whole zebrafish and rainbow trout tissue samples (muscle, liver, gill, intestine) spiked with 100 pg/i TiOz NPs with 2 % Trito X-100 added following acid digestion and measured on ICP-OES calibrated using (a) standards with no added Triton 676 X-100 and (b) standards containing 2 % Triton X-100; Figure 3 shows recovery of Ti in TiQ₂ NP spiked samples containing 2 % Triton X-100 following vortexing of samples for 10 s immediaiely prior to analysis by !CP-OES. (a) Rainbow trout gill and muscle tissue spiked with 200 pg/l, and (b) muscle tissue with 200-1000 pg/f Τ ¾ NP spikes. Data are mean ± SEM, % recovery, n = 6. Different letters indicate significant differences between bars (ANOVA, P < 0.05), with the same letter indicating no significant difference (ANQVA, P > 0.05); and

Figure 4 shows ⁴⁷Ti time scans of (a) Ti metal, (b) bulk TiO_?., (c) Ti0₂ NP standards, and (d) spiked gill tissue digests, all at 200 pg Γ¹ (note the differing maximum count number on the y axis of each panel). ⁴⁷Ti calibration graphs of standards ranging from 0-200 pg I^"1 for (e) Ti metal, (f) bulk Ti0₂, (g) Ti0₂ NP, and (h) Ti0₂ NPs in giil tissue digests. Linear equations and F? values are included for each calibration. Stock rainbow trout (Oncorhynchus mykiss) and zebrafish {Danio rerio) were kept in stock aquaria with flowing, aerated, Plymouth tap water (dechlorinated b standing with aeration for at least 24h prior to use in tanks). The ionic composition of the dechlorinated tap water was 0.3, 0.1 , and 0.4 mmol/l of Na⁺, K⁺ and Ca^2" respectively and the photoperiod for the stock fish was set to a 12h light: 12h dark cycle. The rainbow trout used varied in age from fingerlings to 1 year old juveniles with a wet weight range of 20-900 g wherea the zebrafish were all mature aduiis with a weight range of 0,3-0.9 g (wet weight). For sample collection, fish were terminally anaesthetised with S222 and dissected to harvest target organs using acid cleaned instruments (triple washed in 5 % nitric acid and then triple washed in deionised water). Dissected tissues or whole zebrafish were thoroughly rinsed with deionised water, blotted dr and placed onto new, acid washed, slides in preparation for dehydration.

The titanium dioxide NP powder used herein was from a previously characterised batch (f ederici et at, 2007) and stock solution preparation was identical in the present study. Briefly, dry, powdered TiC½ NPs ("Aeroxide" P25 TJQ₂, DeGussa AG, supplied via Lawrence Industries, Tamworth, UK) comprised 25 % rutile and 75 % anatase TiO_z, had a purity of at feast 99 % Ti0₂ < 1 % Si), an average particle size of 21 nm and a specific surface area of 50 ± 15 m²/g. Chemical analysis of stock solutions revealed no metal impurities (data not shown), and particle analysis by transmission electron microscopy (TEM, JEOL 1200EXH) showed a mean primary particle size of 24.1 ± 2.8 nm (mean ± SEM, n = 100 electron: microscope images, see Federici et ai. 2007). A 10 g/l stock solution of Ti0₂ NPs was generated without solvents by dispersing the NPs in uftrapure Milii-Q (Mifiipore} water with sonication (bath type sonicator, 35 kHz frequency, Fisherbrand FB 1 1010, Germany) for 6 hours.

The starting point for the development of the present invention was an existing method for trace element analysis in fish tissues (Shaw and Handy, 2006). Although spike recovery tests performed using this existing method on tissue samples typically gave recoveries of 100 ± 5 % of the target value for trace elements, the existing method is not suitable for analysing Ti as Ti0₂ NPs. in the existing method, fish tissues were oven dried to constant weight over 48 h (100°C, Gaflenkamp Oven BS Model 1 12 OV- 160), then transferred to 20 mi plastic polypropylene (with polyethylene cap) scintillation vials (VWR International Ltd, Poole, UK) and approximately 0,3-0.1 g of dried tissue was digested in 4 m! of concentrated nitric acid (69 % analytical grade, Fisher Scientific) for 2 h at /O^G in a water bath, removed to cool (for at least 10 minutes), and then diluted to 16 mi using Milli-Q water (resistivity 8.2 Ω cm). For very small tissue samples (less than 0,1 g dry weight) the volumes of reagents were reduced pro rata to a minimum of 1 ml of nitric acid, and diluted to a final volume of 4 ml. Samples were analysed for Ti, Cu, Zn, Mn, Ca, Na and K by inductively coupled plasma optical emission spectrometry (ICP-OES, Varian 725-ES, Melbourne, Australia).

Varian 725 ES ICP-OES operating parameters were; power, 1.4 kW, plasma, auxiliary and nebuliser flows, 15, 1.5, and 0.68 S/min, respectively, and instrument stabilisation, time uptake delay, and replicate read time, 10, 15, and 4 s, respectively, with a wavelength of 336.122 nm for Ti. Calibration was achieved with mixed, matrix-matched standards between 0-1000 mg/l (depending upon anaiyte), prepared from Aristar® plasma emission grade solutions. The calibration blank contained 25 % nitric acid with no standards. Calibration of the ICP-OES for Ti analysis was successfully achieved using either Tt- Metal or Ti0₂ NPs, and for practical reasons the former was used throughout. The: detection limit of the instrument for Ti-Metal (3 x standard deviation of the 25 % nitric acid blank) was 7.04 pg/l {n - 18). The procedural detection limit of Ti-Metal for tissue digests going through the entire protocol (3 x standard deviation of the digestion protocol blank) was 4.58 pg/i (n ~ 6). Titanium dioxide values were calculated from the Ti-Metal values using stoichiometric conversion based on atomic weight In a typical sample run, the blank or a standard was checked (run as a sample) after every 10 samples. In the absence of certified fish reference tissues for total Ti analysis, or for the Ti<¾ NP content of tissues, spike recovery tests using both Ti-Metal and TiO_a NPs were conducted using rainbow trout intestine. Samples were oven dried as described above and known concentrations of Ti-Metal or TiC½ NPs were added to the vial prior to the addition of concentrated nitric acid and the subsequent digestion, Samples were then diluted with Miili-Q water as described above. However, the existing method of Ti analysis, as expected, gave poor results for TiQ₂ NP spiked tissue and it was evident that an alternative method of analysis was required that was optima! for Ti0₂ NPs.

The present applicant has identified that high recoveries of nanoparticles from tissue samples in subsequent analyses by ICP-OES and iCP-AES may be achieved by ensuring that the sample preparation maximises and maintains the dispersion of nanoparticles in the sample. The applicant has further identified that this may be achieved b a combination of: introducing an appropriate surfactant in a concentration selected to maximise dispersion and agitating the sample in an appropriate manner prior to introducing it to the instrument. According to one embodiment of the invention, the following optimised method was developed. For the tissue digestion phase, a non-ionic surfactant, Triton X-100, was added during sample dilution. Tissue samples were processed for acid digestion as described above in the existing method, however once samples were cool, Triton X- 100 was slowly added to each digested sample prior to dilution with Milli-G water to achieve a final voiume of 2 % Triton X-100 in each sample (3.2 or 0.8 ml of the 10 % Triton X-100 solution was carefully pipetted into each digest vial for 16 or 4 ml final dilutions, respectively). Following the addition of Triton X-100 and final dilution (to 16 ml or 4 ml respectively), samples were then stored in cool, dark place until subsequent analysis.

Prior to each analysis, samples were placed on an orbital shaker (KS501 digital orbital shaker, iKA Labortechnik) set at 145 revolutions/mm for a minimum of 30 min to ensure proper mixing of the sample. Samples were sequentially removed from the orbital shaker and immediately analysed by fCF-OES without further agitation for Ti, Gu, Zn, n, Ca, Na and (!CP-OES parameters set precisely as previously described above). It was found that immediately analysing samples after this agitation resulted in marked improvements in recovery of Ti from Ti0₂ NPs. in some embodiments, the tissue sample is placed in a container that maximises the dispersal of nanop rttcfes under agitation. In one embodiment, a 15ml polypropylene copolymer' centrifuge tube with polyethylene cap (Elkay, Basingstoke, UK) is used for digestion and subsequent agitation, in addition or as an alternative to agitation using the orbital shaker the sample may be vortexed immediately prior to analysis, for example for 0 seconds at 2500 r/min, to ensure proper mixing of the sample, It was found that vortexing a sample contained in a scintillation viai was less effective than vortexing a sample in a centrifuge tube. It was found that a combination of the two agitation methods resulted in the best recoveries.

Investigations into matrix matched standards were used (i.e., containing 25 % nitric acid and 2 % Triton X-100), showed that recovery and accuracy was greater when using standards without 2% Triton X- 00 (see Figure 2).

Figure 1 shows the improvements in recovery using the method of the present invention, in which 2% Triton X-100 is added compared with a control method and a further method in which an anionic surfactant, SDS (sodium dodecyl sulphate) was added. The results using 2% Triton X-100 according to an embodiment of the present invention show significantly improved recovery (ANOVA, P<0.05) compared with both the control and SDS. Different letters in Figure 1 indicate statistically significant differences between methods (ANOVA, P<0.05).

The precision of the optimised method was assessed, firstly by comparing within $ampie_: variation (the same trout muscle sample measured in triplicate) in order to ascertain the coefficient of variation of the ICP-OES, and secondly by measuring multiple muscle segments (each as: individual samples) from: the flesh of one fish, to get a measure of the procedural variation. The results are shown below in Table 1. Table : Instrument and procedure precision in rainbow trout muscle and gill tissue spiked with 200. pg/ITidz NPs

Within sample coefficient of variation (CV %) at end of each row is the variation from repeat (x3) measurements of the same tissue sample; CV % at the bottom of a column is the total procedural variation from acid digestion of pieces of muscle from the same fish

Between fish variation was assessed by measuring Ti levels in tissues from different animals. To assess any differences in tissue used, trout gills and muscles were then tested for precision over a serial dilution of Ti0₂ NP Spikes (100-1000 g/l), and the results are shown below in Table 2 and 3. Data for precision in both tables are presented per gram of tissue.

Procedural precision from triplicate digestions of rainbow trout gill tissue at different TiCfe NP spike concentrations

Procedural precision calculated from triplicate digestions of rainbow trout gill tissue at different TiOj spike concentrations. Between sample coefficient of variation (CV%) is the total procedural variation from acid digestions of piece of gill tissue from the same fish Procedural precision from triplicate digestions of rainbow trout muscle tissue at different Τ1Ό2 MP spike concentrations

Procedural precision calculated from triplicate digestions of rainbow trout muscle tissue at different T1O2 spike concentrations. Between sample coefficient of variation (CV%) is the totaS procedural variation from acid digestions of piece of gill tissue from the same fish

Within-sample precision using rainbow trout muscle tissue (triplicate readings from the same sample; Table 1 ) produced coefficient of variation (CV) values ranging from 2.20 to 5,28% in muscle and 0.53 to 6.02 % in gill samples. Within-fish variation (differences when muscle or gill tissue from one fish was divided equally, each piece processed as individual samples and measured for Ti content) was also low (5.53 and 7.68 % for muscle and gill respectively, Table 1).

Gills from six different trout were digested following spiking with a serial dilution of Ti0₂ NP (n - 3 gill samples per fish, Table 2). The variation in the gill tissue was low with all coefficients being under 10 %, Muscle segments from; five different trout were analysed in the same way (Table 3), showing low CV even at increasing concentrations. Although recovery was reduced at increased Ti0₂ NP concentration, variation remained similar throughout.

Samples spiked with 200 pg/i Cu (as CuSO₄,5H₂0) were also analysed to assess instrument and procedural precision and gave within sample CV values of 1 .25, 0.85, 1 .20 and 0.90%, with a within fish CV of 4.58%. A series of spike recovery tests were conducted using a serial dilution of Ti0₂ NPs (0- 1000 μg/i} in order to validate the efficacy and precision of the method. Recovery was tissue and spike concentration dependent with the highest recovery achieved using whole zebrafish and the lowest using liver, whilst 100 pg/i Ti0₂ NP spikes provided the highest recovery in each tissue type compared with more concentrated spikes. Figure 2 shows recovery of 100 pg/l TiQ₂ NP spikes in varying types of tissue and fish. Figure 2(a) shows results obtained using standards that did not include Triton X-1 QG (therefore overestimating recovery), and Figure 2(b) shows results obtained using standards containing Triton X-100.

Figure 3 shows the recovery Ti in Ti0₂ NP spiked samples containing 2 % Triton X-100 following ortexing of samples for 10 s immediately prior to analysis by ICP-OES. (a) Rainbow trout gill and muscle tissue spiked with 200 pg Γ¹, and (b) muscle tissue with 200-1000 pg l^{' !} Ti0₂ NP spikes. Data are mean ± SEM, % recovery, n - 6. Different letters indicate significant differences between bars (ANOVA, P < 0.05), with the same letter indicating no significant difference (ANOVA, P > 0.05).

The effective shelf life of tissue digest samples containing 2 % Triton X-100 was investigated by measuring the samples immediately after 2 hours, 3 and 14 days after the addition of Triton X-100. Notably, 14 days after adding the Triton X-100, Ti recovery from the Ti0₂ NP spiked samples was significantly reduced compared with the initial measurement (e.g. spike recovery of 45 % in intestine samples after 14 d compared to approximately 70 % after 2 h, ANOVA, P < 0.05), whilst an approximately 5 % reduction was noticed after 3 days compared to 2 h. in order to ascertain whether samples containing Triton X-100 could also be used to accurately measure other ana!ytes, samples were tested for differences in tissue Ca, Na, Cu_s Zn, n, and K levels with and without Triton X-100. No significant differences were seen in samples or standards containing 2 % Triton X-100 compared to those without (ANOVA, P>0.05) indicating that interferences were not a concern.

The concentration of Triton X-100 used was much higher that the critical micelle concentration (CMC for Triton X-100 being 0.22 to 0.24 mM in water). The skilled person will appreciate that this would aid the dispersion of NPs in the samples.

Although a method has described in which Triton X-100 is used to aid dispersion of the nano-particles, other non-ionic surfactants may be suitable. The appropriate concentration of other non-ionic surfactants may be different to that found optimal for Triton X-100.

According to another embodiment of the invention, a tissue sample may be analysed ICP-MS. in one embodiment, giii tissue digests containing Q-200pg/i Τί0₂ NPs were analysed by single particle ICP-MS. For comparison, standards produced by serial dilutions of Ti metal (from an Aristar plasma emission grade solution), bulk TiQ₂ (99.7% purity, Acros Organics Belgium) and Ti0₂ NPs (as described previously) were prepared in uitrapure water giving concentrations of 0, 25, 50, 100 and 200 pg/l. All samples and standards comprised 25% nitric acid and 2% Triton X-100 with dilution to the appropriate volume with uitrapure water as described: hereinbefore. A Thermo Fisher Scientific X Series 2 inductively coupled plasma mass spectrometer (ICP-MS, Hemel Hempstead, UK) was used for the single particle characterisation. The ⁴⁷Ti isotope was chosen for analysis following the manufacturers advice due to the reiative (compared, for example, to the more abundant ⁴⁸Ts) lack of interferences (e.g., polyatomic or from isotopes of other elements such as Ca). The sample introduction system consisted of a concentric glass nebuiiser and a PC³ spray chamber assembl cooled to 5 °C, Instrument and data acquisition parameters were as: follows: RF Power = 1400 W, Plasma = 13 I min-1 , Auxiliary = 0.7 l/min, Nebulizer = 0.82 i/min, Sample uptake = 1 ml/min, Points per spectral peak = 1, Sweeps = 1 , Dwelt time = 0,1 ms, Readings per replicate = 4,000 and Integration time - 0.4 s. Each standard and sample were run as a new experiment to standardise time delays in instrument wash time and introduction delays, and for ease of data output.

Figure 4 shows "'Ti time scans of (a) Ti metal, (b) bulk Ti0₂. (c) Ti0₂ NP standards, and (d) spiked gill tissue digests, all at 200 pg Γ¹ (note the differing maximum count number on the y axis of each panel). ⁴ⁱTi calibration graphs of standards ranging from 0-200 pg r¹ for (e) Ti metal, (f) bulk Ti0₂, (g) TiQ₂ NP, and (h) Ti0₂ NPs in gill tissue digests. Linear equations and R values are included for each calibration.

Serial dilutions (0-200 pg Γ¹ ) of Ti0₂ NPs in gill tissue digests, and Ti metal, bulk TiQ₂, and Ti0₂ NPs standards were used to calibrate the ICP-MS. Calibrations were successfully generated, with R² values 0.98 (Fig. 4e-h). Pearson Product Moment Correlation analysis showed that linearity was established for each set with correlation coefficients (and P values in parentheses) of 0.994 (0.0156): 1 (1.00 x 10^"6); 0.996 (3.59 x 10^"4); and 0.994 (5.1 X 10^"4) for the spiked gill tissue digests, Ti metal, bulk TiO_¾ and Ti0₂ NPs standards respectively. The raw data from the IGP-1VSS ^A7Ti time scans are plotted in Fig. 4 (a-d) and show the differences between the 200 pg ¹ Ti metal, bulk Ti0₂ and Ti0₂ NP standards and gili tissue digests; spiked with 200 pg ¹ TiQ₂ NPs, The vast majority of readings in the time scans gave ⁷Ti counts of zero, however there were differences in count readings related to the source of Ti (Fig, 4). For example, Ti metal time scans had a higher percentage of readings in the !ower count bins {u to 30,030), whereas bulk TiC½ counts displayed a higher number and greater spread of count readings further up the count bin scale (up to 90.090). The Ti0₂ NP standards showed a greater number of counts at higher ieveis than the Ti meta!, but fewer than the bulk particle (up to 70, 070), whilst the gill digest samples containing Ti0₂ NPs had the highest spread of all with counts up to 170, 170 (Fig. 4).

The raw data from the ICP-MS ^'''Ti time scans are plotted in Fig. 4 (a-d) and show the differences between the 200 pg ¹ Ti metal, bulk Ti0₂ and Tt0₂ NP standards and giil tissue digests spiked with 200 pg Γ¹ Ti0₂ NPs. The vast majority of readings in the time scans gave ⁴⁷Ti counts of zero, however there were differences in count readings related to the source of Ti (Fig, 4). For example, Ti metal time scans had a higher percentage of readings in the lower count bins (up to 30,030), whereas bulk Ti0₂ counts displayed a higher number and greater spread of count readings further up the count bin scale (up to 90,090). The Ti0₂ NP standards showed a greater number of counts at higher ieveis than the Ti metal, but fewer than the bulk particle (up to 70, 070), whilst the gill digest samples containing TiO_K NPs had the highest spread of all with counts up to 1 0, 1 0 (Fig. 4). The results demonstrate that tissue digests comprising nanopartic!es may be analysed by single particle tCP- S, whe such samples are_: prepared with an appropriate surfactant and subject to agitation to ensure dispersal of the nanoparticles. As a sample is introduced to the instrument, a single NP will be converted into a packet of ions and detected as a single puise (Degueldre et at. 2006; Laborda et al. 201 1 ), with the instrument set up to read large numbers of individual intensit readings within a short dwell time. Samples containing dissolved metals will result in a constant stream of ions passing through the plasma giving a comparatively stable intensity versus time signal ( itrano et a!. 2012). Previously this technique has only been used for samples in pristine conditions (e.g. Ag-NPs in pure water, Laborda et at. 201 1 ). An embodiment of the present invention enables iCP-MS to be carried out on tissue digests containing nanoparticles.

The results obtained here with standards in pristine conditions (Fig. 4e-g) echoed some of the patterns observed by Laborda et a! (201 1 ), but importantly good calibration, with discernible peaks, was also achieved using a serial dilution of TiQ₂ NPs in gill digests (Fig, 4). The instrument was ab!e to detect spikes of ions as the Ti0₂ NPs in both gi!l tissue and water samples were ionized in the plasma torch, with the intensity of these spikes notably different to that seen with Ti metal (Fig. 4). Although the skilled person will appreciate that there is room for optimising the technique, for example by adjusting dwell an total run time, and particle concentration, the data demonstrates that particle number concentration and particle size distribution can in principal be evaluated for tissue digests (by correlation to spike tnienstty), See von der Kammer et al. (2012} for further discussion of this technique and its potential use In characterization of NPs.

Although methods have been described in which fish tissues have been analysed for Ti in Τίθ_ϊ NPs, it will be appreciated that the method ma be applied to analyse other tissues, for example from non-aquatic animals, A number of other modifications and alterations may be made to the arrangements described herein without departing from the scope of the invention, as defined in the appended claims.

An improved method is disclosed herein for detecting Ti from Ti0₂ nanoparticies from tissues, with potential applications in ecotoxicology and food safety. The methods of the present invention yield percentage recoveries of 3-10 fold higher than the initial recoveries using established metal analysis techniques. This method therefore represents a major improvement. ft has been shown that the results obtained using methods according to an embodiment of the present invention are reproducible. This is a requirement in both ecotoxicology research and food safety, where the analytical chemist may often only have one suspect sample to analyse. For ecotoxicology, it is also important to determine the responses of groups of fish, and to understand the within and between fish variability in the analysis, both of which have been shown to be within acceptable limits according to an embodiment of the invention.

The present method allows other analytes (than Ti) to be measured simuftaneously enabling a full trace metal analysis to be carried out. Furthermore, it has been shown that a single sample may be analysed by both ICP-OES and iCP-MS, thereby providing both a quantitative analysis of the concentrations of a variety of trace elements and the characteristics of nanoparticies, such relative size distribution and elemental composition.

Claims

CtA! S;

1. A_: method of preparing a tissue sample for analysis of titanium levels therein, the method comprising;

digesting the tissue sample in acid;

subsequently adding a non-ionic surfactant and diluting the digested tissue with water, wherein the concentration of the non-ionic surfactant is selected to adequately disperse titanium oxide nanoparticles present in the diluted sample; and

agitating the sample immediately prior to analysis to further disperse the nanoparticles.

2. A method of analysing tissue, comprising

preparing a sample using the method of claim 1 ; and

promptly following agitation, analysing the sample by using inductively coupled plasma mass spectrometry to determine the characteristics of titanium oxide nanoparticles in the sample.

3. A method of analysing tissue, comprising:

preparing a sample using the method of claim 1; and

promptly following agitation, analysing the sample by using inductively coupled plasma optical emission spectrometry to determine the concentration of metals including titanium in the form of titanium oxide nanoparticles.

4. A method of analysing tissue, comprising:

preparing a sample using the method of claim 1;

promptly following agitation, analysing the sample by:

using inductively coupled plasma optical emission spectrometry to determine the concentration of metals including titanium in the form of titanium oxide nanoparticles; and

using inductively coupled plasma mass spectrometry to determine the characteristics of titanium oxide nanoparticles in the sample.

5. The method of any preceding claim, wherein the non-sonic surfactant is present in the diluted sample at concentrations: in excess of the critical micelle concentration.

6, The method of any preceding claim, wherein the non-ionic surfactant is Triton X-100.

7. The method of claim 6, wherein the diluted sample has a Triton X-100 concentration of between 1.5% and 3% by volume.

8. The method of any preceding claim, wherein the diluted sample is placed in a container seiected to maximise the dispersion of nanoparticles during agitation.

9. The method of ciaim 8, wherein the container is a centrifuge tube.

10. The method of any preceding claim, wherein agitating the sample comprises swirling the sample on an orbital shaker..

11 , The method of claim 8, wherein the orbital shaker rotates at between 130 and 160 revolutions per minute,

12. The method of any preceding claim, wherein the sample is agitated for at least 30 minutes,

13. The method of any preceding claim, wherein the analysis is carried out within less than 5 minutes of agitation.

14. The method of any preceding claim, wherein agitating comprises vortexing the sample,

15. The method of claim 14, wherein vortexing of the sample is carried out immediately prior to analysing the sample.

16. The method of any preceding claim, wherein the acid is nitric acid.

17, The method of claim 16, wherein the acid -s concentrated nitric acid.

18. The method of any preceding claim, wherein the volumetric ratio of the acid and the diluted sample is between 3 and 6,

19. The method of claim 1 where n the calibration standard comprises titanium metal.

20. The method of any preceding claim, wherein the tissue is fis tissue.